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| | === TIRF and Structured Light Microscopy === | | === TIRF and Structured Light Microscopy === |
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| − | 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. | + | 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 the 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. | | 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. |
Revision as of 18:18, 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 the 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
|