Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Sample amount target: several mg of purified protein
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Sample amount target: 100's ug to several mg of purified protein
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =
• Set ALLOWTOPICCHANGE = CemfacGroup

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Sample amount target: several mg of purified protein
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Sample amount target: 100's ug to several mg of purified protein
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =
• Set ALLOWTOPICCHANGE = CemfacGroup

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Sample amount target: several mg of purified protein
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Sample amount target: 100's ug to several mg of purified protein
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =
• Set ALLOWTOPICCHANGE = CemfacGroup

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Sample amount target: several mg of purified protein
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Sample amount target: 100's ug to several mg of purified protein
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =
• Set ALLOWTOPICCHANGE = CemfacGroup

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =
• Set ALLOWTOPICCHANGE = CemfacGroup

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

More info: Biological structural biology & Principles of TEM

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

More info: Details on beginning an EM project

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

More info: EMIP tutorial for helical reconstruction

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

More info: Negative stain single particle EM

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

More info: ET sample prep considerations

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

More info: FEI Helios 650

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

More info: Overview and resources for CLEM

• Set ALLOWTOPICVIEW =

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein.
Requirements: Need to form a 2D array of particles
Time: months to years
Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex.
Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa
Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years
Resolution: 10Å-50Å

Although single particle techniques can handle samples that are not structurally homogeneous, the higher the purity of the sample in terms of identity and conformation the greater chance meaningful data can be generated. Unless the particle of interest can be readily identified, different proteins or different conformations may be averaged together yielding uninterpretable information or lowering the resolution severely.

To determine the homogeneity of your sample, negative stain single particle EM will be used. This technique will also help track optimization of your sample. For some projects this step may already answer your scientific question. When your sample is optimized, a negative stain 3D reconstruction can be generated. If your sample is amenable, then your sample will be screened using cryo methods to generate a cryoEM 3D reconstruction.

Electron tomography

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm.
Time: months to years
Resolution: 30Å-150Å

Electron tomography can produce 3D reconstructions of heterogeneous samples, such as a cell. However, if the sample is greater than 200nm only a portion of the sample can be imaged. To reconstruct a large volume serial sections of sample must be made and imaged.

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images.
Time: months to years
Resolution: 40Å-400Å

FIB/SEM allows 3D reconstruction of cells and tissues. Although there may be a bit of optimization of the sample for imaging, the main time commitment is data analysis. Processing and annotation of the data may take several months, and requires a solid knowledge of the biochemistry or cell biology of the system of interest.

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous
Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately.
Time: months to years
Resolution range: sub nm to mm

CLEM combines several microscopy techniques and requires the user to have experience in fluorescence microscopy and electron microscopy. This technique requires dedicated resource, personnel and time commitments to complete a project.

• Set ALLOWTOPICVIEW =

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

Electron crystallography and helical reconstruction

Single particle reconstruction

Electron tomography

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Correlative Light and Electron Microcopy (CLEM)

• Set ALLOWTOPICVIEW =

-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the three most common structural biological techniques are given below.

Choosing the right EM technique

EM can provide a unique structural perspective by allowing the determination of a 3D structure of an individual protein to imaging a whole organism. As you begin your project you must decide which EM technique would best suit your needs. Though the different approaches are not mutually exclusive of one another if you are just starting a project it would be best to focus on one primary approach.

This flowchart can help you decide what approaches may yield the most informative results.

Common to all these EM techniques:

• A sample has to be prepared and optimized for EM imaging.
• Several EM images or micrographs of your sample are collected.
• The images are analyzed to correct for imperfections during imaging and ensure the best quality data is used.
• The images are further analyzed and combined to create a reconstruction of your sample.

Electron crystallography and helical reconstruction

Sample purity: Moderate to High biochemical and structural homogeneity - requires isolation of protein. Requirements: Need to form a 2D array of particles Time: months to years Resolution: 2Å-10Å

These two techniques are related to each other though specialized software has been created to analyze the data. One may conceptually treat a helical assembly as a 2D array that is wrapped around to form a tube. High protein purity is preferred although there are rare cases where a conformationally heterogeneous protein of interest is enriched, and separates into a 2D crystal to near identity with a uniform conformation.

Single particle reconstruction

Sample purity: High biochemical and structural homogeneity - requires isolation of protein or complex. Requirements: Need to be able to identify particles of interest, larger the protein the better e.g. >300kDa Time: Negative stain - 2D class averages - weeks to months, 3D reconstruction - months to years; cryoEM - years Resolution: 10Å-50Å

Electron tomography

Sample purity: Biochemically and structurally heterogenous Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Samples cannot be thicker than ~200nm, Time: months to years Resolution: 30Å-150Å

Focus Ion Beam Scanning Electron Microscopy (FIB/SEM)

Sample purity: Biochemically and structurally heterogenous Requirements: Need to be able to identify sample or region of interest from a heterogenous population. Need to optimize sample density and preparation to yield the best quality images. Time: months to years Resolution: 40Å-400Å

Correlative Light and Electron Microcopy (CLEM)

Sample purity: Biochemically and structurally heterogenous Requirements: Need to tag region of interest with a fluorescent marker. Need to be highly familiar with EM to handle samples appropriately. Time: months to years

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-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Ed's Overview of EM (cartoon version)

Choice of an appropriate structural biological technique

Before beginning a project one must match the biological question with the appropriate structural biological technique. A rough guideline for the resolution ranges of the 3 most common structural biological techniques are given below.

subsub level topic

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-- EdEng - 02 Jan 2013

* single particle I:

* single particle II:

* tomography:

* fib sem:

* clem:

Ed's Overview of EM (cartoon version)

subsub level topic

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-- EdEng - 02 Jan 2013

Ed's Overview of EM (cartoon version)

Sublevel topic

subsub level topic

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-- EdEng - 02 Jan 2013