Difference between revisions of "Microscopes"

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= '''Microscopes in Molecular Medicine''' =
+
== '''Microscopes in Molecular Medicine''' ==
  
−
==  Raffi Aroian's Spinning Disk Confocal Microscope ==
+
There are several types of microscopes available with different imaging modalities, different sensitivities and different imaging speeds.
 +
 
 +
=== Spinning Disk Confocal Microscopes ===
 +
 
 +
Some microscopes can improve the optical resolution by using point excitation and emission such as the Spinning Disk Confocal Microscopes. These are usually slower than other microscopes due to the need to scan the point across the specimen.
 +
 
 +
====  Raffi Aroian's Spinning Disk Confocal Microscope ====
  
 
{|
 
{|
Line 54: Line 60:
 
|}
 
|}
  
−
== Stephen Doxsey's Spinning Disk Confocal Microscope ==
+
==== Stephen Doxsey's Spinning Disk Confocal Microscope ====
  
 
{|
 
{|
Line 106: Line 112:
 
|}
 
|}
  
−
== Stephen Doxsey's Epifluorescence Microscope ==
+
=== TIRF and Structured Light Microscopy ===
 +
 
 +
TIRF microscopes work by only exciting fluorophores close to two interfaces with different indexes of refraction, such as a coverslip and aqueous media. At a high incident angle, the laser excitation beam can reflect off the aqueous medium creating an evanescent wave that can penetrate the specimen approximately 200 nm, with an exponential decay. This technique is exceptional at imaging anything near plasma membrane such as vesicle trafficking.
 +
 
 +
Structured Light Microscopy increases spatial resolution by allowing the collection of additional high frequency information by using a shifting illumination pattern. The microscope below leverages structured illumination to determine depth of structures, such as vesicles, in the TIRF field. Due to the exponential decay of illumination, intensity information in TIRF is a based on fluorophore quantity and TIRF depth. Structured Light Microscopy allows one to measure intensity with high fidelity and that measurement can then be used to unconfound fluorophore quantity from TIRF depth. The result can be used to locate fluorescent structures in Z with exceptional accuracy.
 +
 
 +
====  TIRF EpiFluorescence Structured Light Microscope (TESM) ====
  
 
{|
 
{|
 
|Types of Imaging
 
|Types of Imaging
−
|Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
+
|Real time 2D/3D live cell imaging of fluorophores in low to high quantity near or in the plasma membrane.
 
|-
 
|-
 
|Example Fluorophores
 
|Example Fluorophores
Line 116: Line 128:
 
|-
 
|-
 
|Microscope
 
|Microscope
−
|Zeiss Axio Observer D1
+
| Olympus ix71
 
|-
 
|-
 
|Software
 
|Software
−
|Metamorph
+
|[https://micro-manager.org/ μManager]
 
|-
 
|-
 
|Light Sources
 
|Light Sources
−
|X-Cite 120LED (4 Color), Halogen Lamp
+
|Halogen Lamp, Lasers ('''405nm''', 491nm, 561nm, 660nm )
 
|-
 
|-
−
|Wide-field Camera
+
|Camera
−
|Digital CCD Hamamatsu Orca Flash 4, 2048x2048, 6.45&mu;m<sup>2</sup> pixels, '''16 bit''' (2  * 11 bit D/A merged), QE < 82%
+
|Andor iXon 885 Em, 1004x1002,'''14 bits''', 8&mu;m<sup>2</sup>,30fps, QE > 65%
 
|-
 
|-
 
|Differential interference contrast (DIC)
 
|Differential interference contrast (DIC)
−
|Yes
+
|No
 
|-
 
|-
 
|TIRF
 
|TIRF
−
|No
+
|'''Yes'''
 
|-
 
|-
 
|Structured Light
 
|Structured Light
−
|No
+
|'''Yes'''
 
|-
 
|-
 
|Focus Stability
 
|Focus Stability
−
|No
+
|'''Yes ([http://big.umassmed.edu/wiki/index.php/PgFocus pgFocus] by Karl Bellvé)'''
 
|-
 
|-
 
|XYZ Motorized Stage
 
|XYZ Motorized Stage
−
|'''Yes'''
+
|No
 
|-
 
|-
 
|Nano Z Stage
 
|Nano Z Stage
Line 146: Line 158:
 
|-
 
|-
 
|Piezo Z
 
|Piezo Z
−
|No
+
|'''Yes'''
 
|-
 
|-
 
|Microablation System
 
|Microablation System
Line 152: Line 164:
 
|}
 
|}
  
−
== Paul Furcinitti's Epifluorescence Microscope ==
+
=== Epifluorescence Microscopy ===
 +
 
 +
====  Virus Epifluorescence <s>Structured Light</s> Microscope (VESM) ====
  
 
{|
 
{|
 
|Types of Imaging
 
|Types of Imaging
−
|Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
+
|Faster than real time 2D/3D live cell imaging of two fluorophores simultaneously in low to high quantity.
 
|-
 
|-
 
|Example Fluorophores
 
|Example Fluorophores
−
|Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
+
|Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 633, Alexa 632, Cy5
 
|-
 
|-
 
|Microscope
 
|Microscope
−
|Olympus IX70
+
|Olympus ix81
 
|-
 
|-
 
|Software
 
|Software
−
|Metamorph
+
|[https://micro-manager.org/ &mu;Manager]
 
|-
 
|-
 
|Light Sources
 
|Light Sources
−
|Halogen Lamp, Mercury Arc Lamp, Xenon Arc Lamp
+
|Halogen Lamp, X-Cite LED 4 Color ('''395nm''', 470nm, 550nm, 640nm)
 +
|-
 +
|Camera 1
 +
|Andor Zyla CMOS 4.2, '''2048x2048''', '''12 & 16 bit modes''', 6.45&mu;m<sup>2</sup>, '''100fps''', QE >70%
 
|-
 
|-
−
|Wide-field Camera
+
|Camera 2
−
|Roper Scientific Coolsnap HQ, 1392x1040, 6.45&mu;m<sup>2</sup> pixels, 12 bit, QE < 65%
+
|Andor Zyla CMOS 4.2, '''2048x2048''', '''12 & 16 bit modes''', 6.45&mu;m<sup>2</sup>, '''100fps''', QE >70%
 
|-
 
|-
 
|Differential interference contrast (DIC)
 
|Differential interference contrast (DIC)
Line 183: Line 200:
 
|-
 
|-
 
|Focus Stability
 
|Focus Stability
−
|No
+
|'''Soon ([http://big.umassmed.edu/wiki/index.php/PgFocus pgFocus] by Karl Bellvé)'''
 
|-
 
|-
 
|XYZ Motorized Stage
 
|XYZ Motorized Stage
Line 192: Line 209:
 
|-
 
|-
 
|Piezo Z
 
|Piezo Z
−
|Yes
+
|'''Yes'''
 
|-
 
|-
 
|Microablation System
 
|Microablation System
 
|No
 
|No
 +
|-
 +
|Notes
 +
|Housed in a Biosafety Level 2+ Room. Please see [https://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf CDC Biosafety Level Criteria]
 
|}
 
|}
  
−
 
+
==== Stephen Doxsey's Epifluorescence Microscope ====
−
== Virus Epifluorescence <s>Structured Light</s> Microscope (VESM) ==
 
  
 
{|
 
{|
 
|Types of Imaging
 
|Types of Imaging
−
|Faster than real time 2D/3D live cell imaging of two fluorophores simultaneously in low to high quantity.
+
|Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
 
|-
 
|-
 
|Example Fluorophores
 
|Example Fluorophores
−
|Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 633, Alexa 632, Cy5
+
|Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
 
|-
 
|-
 
|Microscope
 
|Microscope
−
|Olympus ix81
+
|Zeiss Axio Observer D1
 
|-
 
|-
 
|Software
 
|Software
−
|[https://micro-manager.org/ &mu;Manager]
+
|Metamorph
 
|-
 
|-
 
|Light Sources
 
|Light Sources
−
|Halogen Lamp, X-Cite LED 4 Color ('''395nm''', 470nm, 550nm, 640nm)
+
|X-Cite 120LED (4 Color), Halogen Lamp
 
|-
 
|-
−
|Camera 1
+
|Wide-field Camera
−
|Andor Zyla CMOS 4.2, '''2048x2048''', '''12 & 16 bit modes''', 6.45&mu;m<sup>2</sup>, '''100fps''', QE >70%
+
|Digital CCD Hamamatsu Orca Flash 4, 2048x2048, 6.45&mu;m<sup>2</sup> pixels, '''16 bit''' (2 * 11 bit D/A merged), QE < 82%
−
|-
 
−
|Camera 2
 
−
|Andor Zyla CMOS 4.2, '''2048x2048''', '''12 & 16 bit modes''', 6.45&mu;m<sup>2</sup>, '''100fps''', QE >70%
 
 
|-
 
|-
 
|Differential interference contrast (DIC)
 
|Differential interference contrast (DIC)
−
|No
+
|Yes
 
|-
 
|-
 
|TIRF
 
|TIRF
Line 233: Line 249:
 
|-
 
|-
 
|Focus Stability
 
|Focus Stability
−
|'''Soon ([http://big.umassmed.edu/wiki/index.php/PgFocus pgFocus] by Karl Bellvé)'''
+
|No
 
|-
 
|-
 
|XYZ Motorized Stage
 
|XYZ Motorized Stage
−
|No
+
|'''Yes'''
 
|-
 
|-
 
|Nano Z Stage
 
|Nano Z Stage
Line 242: Line 258:
 
|-
 
|-
 
|Piezo Z
 
|Piezo Z
−
|'''Yes'''
+
|No
 
|-
 
|-
 
|Microablation System
 
|Microablation System
 
|No
 
|No
−
|-
 
−
|Notes
 
−
|Housed in a Biosafety Level 2+ Room. Please see [https://www.cdc.gov/biosafety/publications/bmbl5/bmbl5_sect_iv.pdf CDC Biosafety Level Criteria]
 
 
|}
 
|}
  
−
== TIRF EpiFluorescence Structured Light Microscope (TESM) ==
+
==== Paul Furcinitti's Epifluorescence Microscope ====
  
 
{|
 
{|
 
|Types of Imaging
 
|Types of Imaging
−
|Real time 2D/3D live cell imaging of fluorophores in low to high quantity near or in the plasma membrane.
+
|Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
 
|-
 
|-
 
|Example Fluorophores
 
|Example Fluorophores
Line 261: Line 274:
 
|-
 
|-
 
|Microscope
 
|Microscope
−
| Olympus ix71
+
|Olympus IX70
 
|-
 
|-
 
|Software
 
|Software
−
|[https://micro-manager.org/ &mu;Manager]
+
|Metamorph
 
|-
 
|-
 
|Light Sources
 
|Light Sources
−
|Halogen Lamp, Lasers ('''405nm''', 491nm, 561nm, 660nm )
+
|Halogen Lamp, Mercury Arc Lamp, Xenon Arc Lamp
 
|-
 
|-
−
|Camera
+
|Wide-field Camera
−
|Andor iXon 885 Em, 1004x1002,'''14 bits''', 8&mu;m<sup>2</sup>,30fps, QE > 65%
+
|Roper Scientific Coolsnap HQ, 1392x1040, 6.45&mu;m<sup>2</sup> pixels, 12 bit, QE < 65%
 
|-
 
|-
 
|Differential interference contrast (DIC)
 
|Differential interference contrast (DIC)
Line 276: Line 289:
 
|-
 
|-
 
|TIRF
 
|TIRF
−
|'''Yes'''
+
|No
 
|-
 
|-
 
|Structured Light
 
|Structured Light
−
|'''Yes'''
+
|No
 
|-
 
|-
 
|Focus Stability
 
|Focus Stability
−
|'''Yes ([http://big.umassmed.edu/wiki/index.php/PgFocus pgFocus] by Karl Bellvé)'''
+
|No
 
|-
 
|-
 
|XYZ Motorized Stage
 
|XYZ Motorized Stage
Line 291: Line 304:
 
|-
 
|-
 
|Piezo Z
 
|Piezo Z
−
|'''Yes'''
+
|Yes
 
|-
 
|-
 
|Microablation System
 
|Microablation System
Line 297: Line 310:
 
|}
 
|}
  
−
==  Multi-Well Microscope ==
+
====  Multi-Well Microscope ====
  
 
{|
 
{|

Revision as of 18:17, 18 January 2017

Microscopes in Molecular Medicine

There are several types of microscopes available with different imaging modalities, different sensitivities and different imaging speeds.

Spinning Disk Confocal Microscopes

Some microscopes can improve the optical resolution by using point excitation and emission such as the Spinning Disk Confocal Microscopes. These are usually slower than other microscopes due to the need to scan the point across the specimen.

Raffi Aroian's Spinning Disk Confocal Microscope

Types of Imaging Real time 2D, with slower 3D, live cell imaging of fluorophores in modest to high quantity
Example Fluorophores Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 633, Alexa 633, Cy5
Microscope Nikon TE2000
Software Metamorph
Light Sources Xeon Arc Lamp, Halogen Lamp, 4 Lasers (405nm, 488nm, 561nm, 636nm)
Wide Field Camera Photometrics CoolSNAP HQ, 1392x1040, 12 bits, 6.45μm2 pixels, QE: 55-62%
Spinning Disk Camera QImaging Rolera-MGi, 512x512, 14 bits, 16μm2 pixels, 30 fps, QE: up to 90%
Nipkow Spinning Disk Yokogawa CSU-10
Differential interference contrast (DIC) Yes
TIRF No
Structured Light No
Focus Stability Yes (Perfect Focus by Nikon)
XYZ Motorized Stage Yes
Nano Z Stage No
Piezo Z No
Microablation System Yes

Stephen Doxsey's Spinning Disk Confocal Microscope

Types of Imaging Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
Example Fluorophores FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
Microscope Zeiss
Software Metamorph
Light Sources Mercury Arc Lamp, Halogen Lamp, 3 Lasers (491nm, 561nm, 647nm)
Wide-field Camera None
Spinning Disk Camera Hamamatsu C4742-80, 1344x1024, 12 bits, 6.45μm2 pixels, 8.8fps, QE : 60-72%
Nipkow Spinning Disk Yokogawa CSU-10
Differential interference contrast (DIC) Unknown
TIRF No
Structured Light No
Focus Stability No
XYZ Motorized Stage Yes
Nano Z Stage No
Piezo Z Yes
Microablation System No

TIRF and Structured Light Microscopy

TIRF microscopes work by only exciting fluorophores close to two interfaces with different indexes of refraction, such as a coverslip and aqueous media. At a high incident angle, the laser excitation beam can reflect off the aqueous medium creating an evanescent wave that can penetrate the specimen approximately 200 nm, with an exponential decay. This technique is exceptional at imaging anything near plasma membrane such as vesicle trafficking.

Structured Light Microscopy increases spatial resolution by allowing the collection of additional high frequency information by using a shifting illumination pattern. The microscope below leverages structured illumination to determine depth of structures, such as vesicles, in the TIRF field. Due to the exponential decay of illumination, intensity information in TIRF is a based on fluorophore quantity and TIRF depth. Structured Light Microscopy allows one to measure intensity with high fidelity and that measurement can then be used to unconfound fluorophore quantity from TIRF depth. The result can be used to locate fluorescent structures in Z with exceptional accuracy.

TIRF EpiFluorescence Structured Light Microscope (TESM)

Types of Imaging Real time 2D/3D live cell imaging of fluorophores in low to high quantity near or in the plasma membrane.
Example Fluorophores Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
Microscope Olympus ix71
Software μManager
Light Sources Halogen Lamp, Lasers (405nm, 491nm, 561nm, 660nm )
Camera Andor iXon 885 Em, 1004x1002,14 bits, 8μm2,30fps, QE > 65%
Differential interference contrast (DIC) No
TIRF Yes
Structured Light Yes
Focus Stability Yes (pgFocus by Karl Bellvé)
XYZ Motorized Stage No
Nano Z Stage No
Piezo Z Yes
Microablation System No

Epifluorescence Microscopy

Virus Epifluorescence Structured Light Microscope (VESM)

Types of Imaging Faster than real time 2D/3D live cell imaging of two fluorophores simultaneously in low to high quantity.
Example Fluorophores Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 633, Alexa 632, Cy5
Microscope Olympus ix81
Software μManager
Light Sources Halogen Lamp, X-Cite LED 4 Color (395nm, 470nm, 550nm, 640nm)
Camera 1 Andor Zyla CMOS 4.2, 2048x2048, 12 & 16 bit modes, 6.45μm2, 100fps, QE >70%
Camera 2 Andor Zyla CMOS 4.2, 2048x2048, 12 & 16 bit modes, 6.45μm2, 100fps, QE >70%
Differential interference contrast (DIC) No
TIRF No
Structured Light No
Focus Stability Soon (pgFocus by Karl Bellvé)
XYZ Motorized Stage No
Nano Z Stage No
Piezo Z Yes
Microablation System No
Notes Housed in a Biosafety Level 2+ Room. Please see CDC Biosafety Level Criteria

Stephen Doxsey's Epifluorescence Microscope

Types of Imaging Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
Example Fluorophores Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
Microscope Zeiss Axio Observer D1
Software Metamorph
Light Sources X-Cite 120LED (4 Color), Halogen Lamp
Wide-field Camera Digital CCD Hamamatsu Orca Flash 4, 2048x2048, 6.45μm2 pixels, 16 bit (2 * 11 bit D/A merged), QE < 82%
Differential interference contrast (DIC) Yes
TIRF No
Structured Light No
Focus Stability No
XYZ Motorized Stage Yes
Nano Z Stage No
Piezo Z No
Microablation System No

Paul Furcinitti's Epifluorescence Microscope

Types of Imaging Slower than real time 2D/3D imaging of fluorophores in modest to high quantity.
Example Fluorophores Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
Microscope Olympus IX70
Software Metamorph
Light Sources Halogen Lamp, Mercury Arc Lamp, Xenon Arc Lamp
Wide-field Camera Roper Scientific Coolsnap HQ, 1392x1040, 6.45μm2 pixels, 12 bit, QE < 65%
Differential interference contrast (DIC) No
TIRF No
Structured Light No
Focus Stability No
XYZ Motorized Stage No
Nano Z Stage No
Piezo Z Yes
Microablation System No

Multi-Well Microscope

Types of Imaging Slow 2D/3D live cell imaging of fluorophores in high quantity optionally using 96 well plates.
Common Fluorophores Hoechst, DAPI, FITC, GFP, RFP, Cy3, Cy5
Microscope Zeiss Axio Observer Z1
Software μManager
Light Sources Halogen Lamp, Xenon Arc Lamp
Camera Andor Clara, 1392x1040, 14 & 16 bit, 6.45μm2, 11fps, QE 40-60%
Differential interference contrast (DIC) No
TIRF No
Structured Light No
Focus Stability Yes (Definite Focus by Zeiss)
XYZ Motorized Stage Yes
Nano Z Stage No
Piezo Z No
Microablation System No
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