Microscopes

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Microscopes in Molecular Medicine

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

All the images from these microscopes can still benefit from proper processing, analysis and 3D Deconvolution such as the deconvolution software developed by the Biomedical Imaging Group.

Spinning Disk Confocal Microscopy

Some microscopes can improve optical resolution by using point excitation and emission as seen in Confocal Microscopes. These are usually slower than other microscopes due to the need to scan the point across the specimen but the microscopes below use a spinning disk with multiple pinholes to improve time resolution and optical resolution.

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
Type Inverted
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
Type Inverted
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
Type Inverted and Upright
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

These microscopes are designed to excite and image fluorophores. These are your basic workhorse microscopes.

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
Type Inverted
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 Future
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
Type Inverted
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

Epifluorescence Microscope (formerly of Paul Furcinitti's DISC core)

Types of Imaging Slower than real time 2D/3D imaging of fluorophores in modest to high quantity, including 340/380 calcium ratiometric imaging.
Example Fluorophores Fura-2, Hoechst, DAPI, FITC, DyLight 488, Alexa 488, GFP, DyLight 550, Alexa 555, RFP, Cy3, DyLight 650, Alexa 647, Cy5
Microscope Olympus IX70
Type Inverted
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 Epifluorescence 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
Type Inverted
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

Simple Microscopy

Zeiss Microscope

Types of Imaging Brightfield
Common Fluorophores
Microscope Zeiss Axioplan 2
Type Upright
Software None
Light Sources Halogen Lamp, Mercury Arc Lamp
Camera None
Differential interference contrast (DIC) No
TIRF No
Structured Light No
Focus Stability No
XYZ Motorized Stage No
Nano Z Stage No
Piezo Z No
Microablation System No

Microscope Scheduling

Microscopes in Stephen Doxsey's group can be scheduled through his lab via a paper signup sheet. The remaining microscopes can be scheduled through the Biomedical Imaging Group, and their online calendaring system after they have establish an account for you.

Microscope Training

New users will be trained in the use and care of microscopes.

Microscope Care

Extreme care needs to be taken around any optical surface and optical fibers, as well as the objectives. Excessive oil can damage objective's seals causing unwanted oil accumulation on optical surfaces below or inside the objective. For example, below is an image of a DIC prism from our Nikon Spinning Disk Confocal. You can see oil accumulation on this prism, and this sits inches below the objective! One only needs to use enough oil to cover the front lens of an objective and the oil must be cleaned from the objective after you are finished. For deep cleaning, we use 85% Petroleum Ether with 15% Isopropanol but an ethanol solution will work as well. Only use lens tissue or cotton swabs once. Clean in a circular motion working from inside to outside.

Oil on DIC Prism

Help

The Biomedical Imaging Group provides expertise in image processing, image analysis, image visualization and 3D Deconvolution.