Resources & Services

 What it’s good for

• Super Resolution Confocality
• Tunable excitation source for hand picking excitation wavelengths of up to 8 simultaneously
• Determining colocalization for multiple signals
• Acquisition of up to 5 color channels simultaneously, all HyDs
• Acquisition of multiple color channels sequentially
• Acquisition of a transmitted light image with DIC
• Fixed cells or tissues on a slide with a coverslip (#1.5)
• Spectral Deconvolution to correct for overlapping signals
• On the fly Lightning image deconvolution for super resolution of 120nm in X and Y and 200nm in Z
• TauSense tools for fast FLIM and Gating

What it’s not good for

• Live Cells: This system does not have incubation for live cell applications
• Samples in a dish or plate

Principles of operation

Laser light of specific wavelengths is scanned across the sample and filtered before detection to produce a high resolution image composed of a small optical slice of the sample. Deconvolution further enhances resolution.

Technical Information

White Light Laser (470nm - 790nm)

• 5x (.15 NA)
• 10x (.40 NA)
• 20x Oil/Water/Glycerol (.75 NA)
• 20x (.40 NA)
• 40x Oil (1.30 NA)
• 63x Oil (1.40 NA)

What it’s good for

• Super Resolution Confocality
• Determining colocalization for multiple signals
• Acquisition of up to 4 color channels simultaneously
• Acquisition of multiple color channels sequentially
• Acquisition of a transmitted light image with DIC
• Fixed cells or tissues on a slide with a coverslip (#1.5) or in a dish
• Spectral Deconvolution to correct for overlapping signals
• On the fly image deconvolution for super resolution of 120nm in X and Y and 200nm in Z

What it’s not good for

• Live Cells: This system does not have incubation for live cell applications

Principles of operation

Laser light of specific wavelengths is scanned across the sample and filtered before detection to produce a high resolution image composed of a small optical slice of the sample. Deconvolution further enhances resolution.

Technical information

• Microscope: Leica DMi8 Inverted
• Spectral for all channels laser lines
• 405nm
• 488nm
• 552nm
• 638nm

Microscope objectives

• 10x (.40 NA)
• 20x (.75 NA)
• 40x Oil (1.30 NA)
• 63x Oil (1.40 NA)

Software & supplemental information

Leica LAS Lite Software Download

Leica Microsystems - WhitePaper LIGHTNING: Image Information Extraction by Adaptive Deconvolution (PDF)

What it’s good for

• STED Super Resolution <50nm in XY
• Tunable excitation source for hand picking excitation wavelengths of up to 8 simultaneously (470nm-670nm)
• FLIM acquisition with Falcon module
• Incubation for live cell imaging (only BSL1 cell lines)
• Determining colocalization for multiple signals
• Acquisition of up to 5 color channels simultaneously
• Acquisition of multiple color channels sequentially
• Acquisition of a transmitted light image with DIC
• Fixed cells or tissues on a slide with a coverslip (#1.5) or in a dish
• Spectral Deconvolution to correct for overlapping signals
• On the fly image deconvolution for super resolution of 120nm in X and Y and 200nm in Z

Principles of operation

Laser light of specific wavelengths is scanned across the sample and filtered before detection to produce a high resolution image composed of a small optical slice of the sample. STED enahances resolution by shrinking fluorescence to a small spot with a depletion laser. Deconvolution further enhances resolution, including STED images.

Technical information

• Microscope: Leica DMi8 Inverted
• Spectral for all channels
• PMT and HyD detectors
• STED - Super Resolution depletion lines
• White Light Laser tunable to any desired excitation between 470 and 670nm

Microscope objectives

• 10x (.40 NA)
• 20x (.75 NA)
• 40x Water (1.10 NA)
• 40x Oil (1.30 NA)
• 63x Oil (1.40 NA)
• 100x Oil (STED)(1.40 NA)

Sample preparation guidelines For STED imaging only:

Choice of samples:

• For Live samples: Media should be clear and contain no phenol red or other color additives
• For Fixed Samples: RI of mounting medium should match RI of immersion used (prolong glass or diamond)
• DAPI should be AVOIDED, especially when using green fluorophores
• Autofluorescence should be extremely low
• It's important that the sample doesn't absorb 592nm, 660nm or 775nm
• Use only #1.5 coverglass, including for glass bottom dishes

Recommended Dyes:

• Single color for 592nm depletion line
  • DyLight 488 or 514
  • Oregon Green 488 or 514
  • AlexaFluor 488 or 514
  • ATTO 488 or 514
• Single color for 660nm depletion line
  • Alexa 532
  • ATTO 532 or 550
  • TMR/TRITC
  • Cy3
  • Alexa 555
• Single color for 775nm depletion line
  • ATTO 647N
  • Alexa 633
  • Alexa 594
  • ATTO 590

Recommended Fluorescent Proteins:

• eGFP (484nm ex / 592nm depletion)
• EmGFP (487nm ex / 592nm depletion)
• eYFP (514nm ex / 592/660nm depletion)
• Venus (515nm ex / 592/660nm depletion)
• mCitrine (516nm ex / 592/660nm depletion)
• dsRed (558nm ex / 660nm depletion)
• mStrawberry (574nm ex / 660nm depletion)
• mKate2 (588nm ex / 775nm depletion)
• Fluorescent Proteins to AVOID: mCherry, CFP, tagRFP

Software & supplemental information

Leica LAS Lite Software Download
STED Sample preparation guide - Confocal Application Letter (PDF)
STED Sample Prep Oct 18 (PPTX)
LIGHTNING WhitePaper (PDF)
STED 3X Sample Prep 2018.pdf - Powered by Box.html

Abberiror Sample Prep guide - (PDF)

What it's good for

• Generating brightfield and multi-color fluorescent images of entire slides or tissue sections automatically
• 7 fluorescent colors
• Fixed cells or sections on a standard 1 x 3 slide with a coverslip (#1.5) or 2 x 3 slides
• Can scan up to 210 slides automatically

What it's NOT good for

• Thick tissue slices (greater than 50um)
• Timelapse or live cell applications

Principles of operation

Fluorescent light from an LED lamp filtered for emission or brighfield light produce an image by tiling specified areas of a slide to generate montage reconstructions.

Technical information

• 7 fluorescent color channels and brightfield:
• DAPI - Ex 345 Em 432/36
• FITC - Ex 494 Em 515/30
• TRITC - Ex 555 Em 600/31
• CY5 - Ex 625 Em 685/42
• Cy7 - Ex 743 Em 810/81
• CFP - Ex 458 Em 482/25
• YFP - Ex 513 Em 544/25
• Motorized X-Y capability for slide scanning
• Microscope objective:
• 2x, 4x, 20x (.80 NA), 40x oil (1.40NA)
• 40x oil lens is for high resolution scans of small areas only. Cannot be used to scan whole slides or for more than one slide at a time.

Files must be named in the proper format to open. There is a folder and thumbnail for each image.

Example

For slide preparations

• No wet slides - must be completely dry
• Use #1 or #1.5 coverslips

Software for Processing

Download OlyVIA Viewer

https://qupath.github.io/

What it's good for

• Generating multi-color fluorescent images 
• Live cell imaging - incubation provided
• Super resolution down to 60nm XY
• 4 fluorescent colors - 2 channel simultaneous acquisition (see table)
• Fixed cells or sections on a standard 1 x 3 slide with a coverslip (#1.5)
• Live cells in a 35mm glass bottom dish, glass bottom chamber slide, or glass bottom plate
• Apotome imaging
• Lattice structured illumination imaging
• Laser wide-field imaging
• TIRF Imaging

What it's NOT good for

• Thick tissue slices (greater than 100um)
• Lattice SIM is not good for samples with diffuse structureless fluorescence or low contrast

Principles of operation

Fluorescent light from lasers filtered for emission produce an image from wide-field resolutions to super resolutions down to 60nm XY. A lattice pattern is placed in the light path to surpass the diffraction limit and produce super resolutions in lattice SIM. A lattice pattern is placed in the light path to improve resolutions over wide-field.

Technical information

• 4 fluorescent color channels:
• DAPI (405nm)
• FITC  (488nm)
• TRITC (561nm)
• CY5 (642nm)
• Motorized X-Y capability for multiposition imaging
• Microscope objectives:
• 10x (.75NA), 20x (.80 NA), 40x (1.40NA water), 63x (1.40NA oil), 100x (1.45NA oil)

Dual Camera Acquisition Parameters: 

For sample preparations

• No wet slides - must be completely dry
• Use #1.5 coverslips or #1.5 coverslip bottom formats

All live cell imaging must be approved by core. Please submit a BUA authorization form along with your lab's BUA report for authorization.

Important Considerations:

• Processing is required when performing Lattice SIM
• A second computer is available for all necessary processing such as SIM2 and Channel Alignment
• Daily alignment of the cameras is required for dual camera acquisition (acquiring two channels simultaneously)
• Alignment of the cameras is not required for single camera use or single fluorophore
• Daily Channel Alignment is required to correct for chromatic shift in XYZ and Rotation when examining more than one fluorophore. To learn about chromatic shift and why it needs to be calibratated the system, please visit:

YouTube Chromatic Shift

This system is capable of performing the following techniques:

• SEM with high pressure and variable pressure modes
• TEM on grids with STEM detector and 12 grid specimen holder
• Gatan 3View Serial Block Face Scanning Electron Microscopy Demonstration (youtube.com)
• ATLAS software large scale acquisitions of TEM grids and Array Tomography sections
• Correlative Microscopy of Light and Electron microscopy techniques
• Array Tomography with SEM

Principles of operation

Serial block face scanning electron microscopy generates EM resolution 3D images. The system has an ultramicrotome inside the chamber of the SEM, which allows for automatic cutting of a tissue block. The surface of the block is imaged by detection of back-scattered electrons and then a thin section (30-50nm) is cut from the block face. The sample block is then imaged again and a sequence of images can be compiled automatically.

For more information, or access to this technology, contact Jennifer Santini.

Gatan 3View System Publications

Probes for Correlative EM

What it’s good for

• Thick specimens and whole organisms: Cleared tissue, zebrafish etc.
• Fast acquisition speeds and low phototoxicity
• Incubation for live imaging
• Acquisition of up to 2 color channels simultaneously
• Acquisition of multiple color channels sequentially

What it's not good for

• Fixed cells or tissues on a slide with a coverslip (#1.5) or in a dish

Principles of operation

Laser light of specific wavelengths is passed through the sample as a sheet from 1 or 2 sides, perpendicular to the detection optics. Laser excitation, Emission filters and 2 CMOS cameras generate an image at a specific wavelength. Sample is moved through the sheet of light for Z stack acquisition.

Technical information

• Filter based emission for all channels
• 2 pico.edge CMOS cameras
• 3 specimen chambers for different objectives and refractive index
• Incubation for chamber
• Laser Excitations:
• 405nm
• 445nm
• 488nm
• 514nm
• 560nm
• 638nm

Microscope objectives

• EC Plan-Neofluar 5x (.16NA) Air
• CLR Plan-Neofluar 20x Corr (1.0NA) dipping
• Plan-Apochromatic 10x (.5NA) dipping
• W Plan-Apochromatic 20x (1.0NA) dipping

What it’s good for

• High resolution imaging at 1.7x resolution increase over standard confocal imaging
• High acquisition speed, at 4x faster acquisition than confocal mode
• High sensitivity with use of special detectors
• Shuttle and Find capability to merge light and electron microscopy techniques for Correlative Microscopy
• Acquisition of up to 3 color channels simultaneously
• Acquisition of multiple channels sequentially
• Acquisition of a high contrast DIC image
• Fixed cells or tissues on a slide with a coverslip (#1.5)
• Live cells, small organisms or tissues in a chamber or dish with a coverslip bottom (#1.5) (only BSL1 cell lines)
• Spectral Deconvolution to correct for overlapping signals
• Calcium Dynamics
• FRET
• Stimulus and bleaching applications

What it’s not good for

• Live animal imaging (large animals such as mouse)

Principles of operation

Laser light of specific wavelengths is scanned across the sample and filtered before detection to produce a high resolution image composed of a small optical slice of the sample. Alternative detection at high resolution and sensitivity avoids discrimination of light with a pinhole, and utliizes a 32 detector array that can generate higher resolution images by detection of specific airy units.

Technical information

• Microscope: Ziess Observer inverted stand
• Incubated with CO2
• DIC channel
• laser lines
• 405
• 458
• 488
• 515
• 543
• 594
• 633

MDPI photonics: Exploring the Potential of Airyscan Microscopy for Live Cell Imaging

Zeiss stage inserts

The Hamamatsu Nanozoomer is an older system. Therefore we no longer perform training sessions on this microscope. Our new Olympus VS200 slide scanner is the replacement system for the nanozoomer. Please request training on the Olympus VS200 slide scanner if you want access to slide scanning technology.

What it's good for

• Generating brightfield images of entire slides or tissue sections automatically
• Fixed cells or sections on a standard 1 x 3 slide with a coverslip (#1.5)
• Can scan up to 210 slides automatically

What it's NOT good for

• Thick tissue slices
• Timelapse

Principles of operation

Fluorescent light from a mercury lamp is filtered for excitation and emission or brighfield light produce an image by scanning specified areas of a slide to generate montage reconstructions.

Technical information

• Motorized X-Y capability for slide scanning
• Microscope objective:
• 20x (.75 NA)

For slide preparations

• No frosted glass
• No beveled edges of any kind on the slides
• No label or coverslips hanging off edge or extra mounting medium on edges
• No wet slides - must be completely dry
• Use #1 coverslips

Software

Hamamatsu Nanozoomer NDP View 2 Software Download

What it's good for

• Low magnification observation
• Brightfield (color)
• Darkfield
• Fluorescence
• Obtaining images of:
• intact organs
• whole sections
• Field view from 55mm to 1.74mm

What it's NOT good for

• High magnification
• Multipoint timelapse
• Low contrast brightfield samples
• Low fluorescence

Principles of operation

Fluorescent light from a mercury lamp is filtered for excitation and emission.

Technical information

• Microscope Base: MVX10
• Color CCD camera
• 4 fluorescent color channels:
• DAPI
• FITC
• Texas Red
• Cy5.5
• Color brightfield
• Microscope Objectives:
• .63x
• 2x