Confocal, Super-Resolution and Digital Light-Sheet Microscopes

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Leica SP8 STED and MP


The Leica SP8 spectral confocal microscope is equipped with three high sensitivity, low-noise hybrid detectors capable of photon counting and two conventional PMT detectors. Sample excitation can be accomplished by multiple lasers including a pulsed white light laser tunable from 470 to 670 nm that allows selection of the optimal excitation wavelength; the LightGate panchromatic electronic barrier filter allows gating of the detectors to avoid collecting signal during the laser pulse so that only pure emission signals are collected. The system also has conventional fixed laser excitation lines at 405, 458, 476, 488, 496 and 514 nm. For two-photon imaging, including FLIM-FRET, the system has a MaiTai DeepSee Ti-Sapphire ultrafast laser (tunable between 680–1060 nm). The SP8 is equipped with both a conventional point scanner for work requiring the highest sensitivity and a resonant scanner for live cell imaging (28 fps at 512x512). An environmental chamber for temperature and CO2 control during prolonged live-cell imaging experiments is available. Complex experimental designs, 3D image tiling and 3D rendering and measurements are possible with the LAS AF Leica software.

The Leica confocal microscope has been upgraded with a 3D STED super-resolution system. This system can achieve a resolution of 50 nm or less. Three depletion lasers (592, 660 and 775 nm) enable multicolor detection of a wide range of fluorophores. Use of the Huygens deconvolution software programs allows for further improved resolution.

Download an STED technology basics and sample preparation guide (PDF).

The following objectives are available for this system:

Mag. Objective Description NA Immersion Medium Working distance (mm)
10x HC PL APO CS 0.40 Air 2.20
20x HC PL APO CS 0.70 Air 0.59
20x HC PL APO IMM CORR CS2 0.75 Water, glycerol, or oil 0.68
25x HCX IRAPO L 0.95 Water 2.40
40x HC PL APO CS2 1.10 Water 0.65
40x HC PL APO CS2 1.30 Oil 0.24
63x HC PL APO CS2 1.40 Oil 0.14
63x HC PL APO CORR CS2 1.30 Glycerol 0.30
63x HC PL APO CORR CS2 1.20 Water 0.30
100x HCX PL APO CORR CS 1.44 Oil 0.10
100x HC PL APO STED WHITE 1.40 Oil 0.13


Download the free Leica LAS X Core Offline software. Scroll down to Downloads.

Leica GSD 3D

The Leica GSD 3D super-resolution microscope with TIRF possesses four different laser lines (488, 560, 642 and 405 nm) and a fast, highly sensitive Andor iXon Ultra 897 EMCCD camera for detecting up to three fluorophores. The microscope also includes a fully automated TIRF system allowing super-resolution to be combined with TIRF microscopy.

Download the GSD miscroscopy basics and sample preparation protocol (PDF).

The following objectives are available for use with the GSD system:

Mag. Objective Description NA Immersion Medium Working distance (mm)
10x HCX PL FLUOTAR 0.30 Air 11
100x HC PL APO CORR TIRF 1.47 Oil 0.09
160x HC PL APO 1.43 Oil 0.07

 

Leica TCS SP8 with digital light-sheet and diode lasers

The Leica TCS SP8 system possesses fixed diode laser lines (405, 488, 552, 638 nm) that can be utilized for conventional confocal microscopy or digital light-sheet applications. Illumination with the light sheet allows rapid acquisition of multiple images (>100 frames/second) to permit 3D rendering and results in low phototoxicity. Imaging can be performed for entire organs (e.g., Arabidopsis roots) or organisms (e.g., Drosophila embryos, developing zebra fish). Extended time-lapse acquisition can be used to monitor physiological or developmental changes over time.

Download the guide to light-sheet microscopy basics and sample preparation protocol (PDF).

The following objectives are available for use with the confocal and light-sheet systems:

Mag. Objective Description NA Immersion Medium Working distance (mm)
10x HC PL APO CS 0.40 Air 2.20
20x HC PL APO CS 0.70 Air 0.59
20x HC PL APO IMM CORR CS2 0.75 Water, glycerol, or oil 0.68
25x HCX IRAPO L 0.95 Water 2.40
40x HC PL APO CS2 1.10 Water 0.65
40x HC PL APO CS2 1.30 Oil 0.24
63x HC PL APO CS2 1.40 Oil 0.14
63x HC PL APO CORR CS2 1.30 Glycerol 0.30
63x HC PL APO CORR CS2 1.20 Water 0.30
100x HCX PL APO CORR CS 1.44 Oil 0.10
100x HC PL APO STED WHITE 1.40 Oil 0.13

 

The following objectives are specifically for use with the light-sheet system:

Mag. Objective Description NA Immersion Medium Working distance (mm)
2.5x HC PL FLUOTAR 0.07 Air 9.4
10x HC APO DLS L 0.30 Water 3.6
25x HC PL APO IMM CORR CS2 0.75 Water 2.5

 

 

Widefield Microscopes

Zeiss Axiovert 200M with Leica DFC290 color camera

The Zeiss Axiovert 200M inverted microscope is a fully automated instrument with a motorized stage and z-drive. This microscope is capable of brightfield and DIC. A featured technique on this system is slide scanning (the automated acquisition of images across a defined region), with the possibility of acquiring high-quality composite images from whole-tissue slides. A wide range of low and high magnification objectives are available. The selection of cameras includes a Leica DFC290 color camera. An environmental chamber with temperature and CO2 control is available for extended imaging of live cells. The system operates under control of the MetaMorph software v.7.8.12.

The following objectives are available for this system:

Mag. Objective Description NA Immersion Medium Working distance (mm) Application
5x Plan Apochromat 0.16 Air 12.1 BF
10x EC Plan-NeoFluar 0.3 Air 5.6 BF, DIC II
20x Plan-Apochromat 0.75 Air 0.61 BF, DIC II
20x Plan-Apochromat 0.8 Air 0.55 BF, DIC II
25x LCI Plan-NeoFluar 0.8 Water/glycerol 0.21 BF
40x LD Plan-NeoFluar 0.6 Air 2.5-3.3 BF
40x EC Plan-NeoFluar 1.3 Oil 0.21 BF, DIC III
40x C-Apochromat 1.2 Water 0.29 BF, DIC III
40x C-Apochromat 1.2 Water 0.28 BF, DIC III
63x Plan-Apochromat 1.4 Oil 0.18 BF, DIC III
63x Plan-Apochromat DIC 1.4 Oil 0.18 BF, DIC III
63x C-Apochromat 1.2 Water 0.28 BF

 

BF-bright field, DIC-differential interference contrast

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Zeiss Axiovert 200M with fluorescence and ORCA-ER camera


The Zeiss Axiovert 200M inverted microscope is a fully automated instrument with a motorized stage and z-drive. A featured technique on this system is fluorescent microscopy; however, the system is also capable of transmitted light bright-field. In addition, the microscope can acquire high-quality composite images of whole-tissue slices (a process termed "tiling") for both single- and multi-channel fluorescence. A wide range of objectives and fluorescence filter sets are available. Images are acquired by a Hamamatsu ORCA-ER CCD camera, a high sensitivity, low-noise device for high resolution imaging. An environmental chamber with temperature and CO2 control is available for extended imaging of live cells. The system operates under the control of the MetaMorph v.7.8.12 software program. This program enables complex experimental designs, including time-lapse imaging, autofocusing, streaming and z-stack acquisition.

The following objectives are available for this system:

Mag. Objective Description NA Immersion Medium Working distance (mm) Pixel size (um) Field of view
5x Plan Apochromat 0.16 Air 12.1    
10x EC Plan-NeoFluar 0.3 Air 5.6 0.9958 1338.4 x 1019.7
20x Plan-Apochromat 0.75 Air 0.61    
20x Plan-Apochromat 0.8 Air 0.55 0.4959 666.4 x 507.8
25x LCI Plan-NeoFluar 0.8 Water/glycerol 0.21 0.3947 530.5 x 404.2
40x LD Plan-NeoFluar 0.6 Air 2.5-3.3 0.2490 334.7 x 255.0
40x EC Plan-NeoFluar 1.3 Oil 0.21 0.2490 334.7 x 255.0
40x C-Apochromat 1.2 Water 0.29    
40x C-Apochromat 1.2 Water 0.28 0.2454 330.0 x 251.3
63x Plan-Apochromat 1.4 Oil 0.18 0.1569 210.9 x 160.7
63x Plan-Apochromat DIC 1.4 Oil 0.18 0.1569 210.9 x 160.7
63x C-Apochromat 1.2 Water 0.28 0.1576 211.8 x 161.4

 

The following fluorescent filters are available for this microscope:

Fluor Filter Set Excitation Emission
DAPI 49000 325-375 435-485
DAPI LP FS2 365 LP420
CFP FS 47 426-446 460-500
eGFP/FITC 49002 450-490 500-550
YFP FS 46 490-510 520-550
Cy3/TRITC 49004 532-557 570-640
DSRed 49005 530-560 590-650
Cy5 49006 590-650 662-737
Cy7 49007 642-747 765-855

 

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Leica DM 5500B Compound Microscope with Leica DFC290 Color Digital Camera


The Leica DM5500 upright microscope is an automated instrument with a motorized z-drive and fluorescence filter wheel, and automatic selection of appropriate optical components for every featured microscopy modality. The microscope is capable of transmitted light brightfield, dark field, phase contrast, DIC and fluorescence imaging. Images can be captured with a Leica DFC290 high resolution color camera or a Leica DFC340 high sensitivity cooled monochrome camera. The system features a fully integrated deconvolution module with 3D Blind, 3D Non-Blind, Golds and inverse filter deconvolution algorithms.

The following objectives are available for this system:

Mag. Objective Description NA Immersion Medium Working distance (mm)
5x N PLAN PH 0 0.12 Air 14.0
10x HC PL FLUOTAR 0.30 Air 11.0
20x HCX PL FLUOTAR 0.50 Air 1.15
40x HCX PL FLUOTAR 0.75 Air 0.40
40x HC PL APO CS2 0.13 Oil 0.24
63x HCX PL APO 1.40 Oil 0.10
63x HC PL APO CS2 CORR 1.20 Water 0.30
100x HCX PL APO CS CORR 1.44 Oil 0.10

 

The following fluorescent filter cubes are available for the Leica DM 5500B microscope:

Filter Cube Excitation range Dichromatic mirror Suppression filter
A4 UV 400 BP 470/40
L5 Green 505 BP 527/30
N3 Blue 565 BP 640/40
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Leica M205 FA stereomicroscope


The Leica M205 FA stereo microscope is a fully automated instrument with a motorized z-drive, focus, zoom and filter wheel controls for multichannel time-lapse experiments. The available filter sets include DAPI, CFP, GFP, YFP and dsRed. The featured objective has a zoom range of 20.5:1 and a working distance of 61.5 mm. The system includes a transmitted light base as well as an O-ring LED illuminator with 48 LEDs. Images are captured with a 2.8 Mpixel Leica DFC7000T color, high-sensitivity, cooled digital camera.

Some special features include Automontage software for extended depth of focus imaging and Rotterman's relief contrast to visualize unstained transparent objects. The system also features a PhotoFluor II illuminator which is an adjustable fluorescence light source providing a very stable high-power output essential for time-lapse and quantitative fluorescent imaging.

Montage image below taken by Michele Warmund. Right image is the multifocus image, and left image is one of the single images in the stack.

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Fluorescent filter example
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Montage group


The following fluorescent filters are available for the Leica M205 FA stereo microscope:

More example montage images (Michele Warmund):
 

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Montage group 1
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Montage group 2

Leica DMI4000B

An inverted, fully automated Leica DMI4000 B with integrated Eppendorf Microinjection system and a 1.4 megapixel, 12-bit, cooled CCD camera from Q-Imaging. This system is capable of injecting a single cell with volumes as small as a femtoliter. The capture software from Q-Imaging allows for quick and easy collection of images for traditional microscopy applications as well.

This system is equipped with four objective lenses:

Mag. Objective Description NA Immersion Medium μm per pixel Field of View (μm) Working Distance(mm) Applications*
5x N Plan 0.12 Air 1.85874 2587.36 x 1933.09 14 BF, FL, Ph0, DIC
10x N Plan 0.25 Air 0.945017 1315.46 x 982.82 5.8 BF, FL, Ph1, DIC
20x N Plan Corr 0.4 Air 0.464134 646.07 x 482.69 3.2-1.9 BF, FL, Ph1, DIC
40x HCX PL Fluotar L Corr 0.6 Air 0.229084 318.88 x 238.25 3.3-1.9 BF, FL, Ph2, DIC

 

The following filters are available on this system:

The following are example images taken by MU researchers:

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Diatomes imaged using DIC

Diatomes imaged using DIC (Differential Interference Contrast) for enhanced details

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Diatomes imaged using Phase Contrast

Diatomes imaged using Phase Contrast, a technique different than DIC, but also for enhancing certain details.

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cell being injected

A cell being injected

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nuclear transfer in an oocyte

Nuclear transfer in an oocyte. Taken by Ye Yuan

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Leica MZ FLIII stereomicroscope


A Leica stereoscope equipped with epi-fluorescence excitation and Chroma HiQ fluorescence filters is also available.

The following Chroma filters are available for the Leica Stereoscope:

 

Laser Capture Mirodissection

ArcturusXT laser capture


An automated system for UV laser cutting (microdissection) and IR laser capture of tissue samples, mounted on a Nikon Eclipse TE2000 inverted microscope. Capable of brightfield, phase-contrast, DIC and fluorescence microscopy. Equipped with a color CCD camera.

Lenses

This system is equipped with five objective lenses:

Mag. Objective Description NA
2x Plan UW 0.06
4x Plan Fluor PhL DL 0.13
10x Plan Fluor Ph1 DL 0.30
40x ELWD Plan Fluor Ph corr. 0.60
60x ELWD Plan Fluor Ph corr. 0.70

 

Filters

The following fluorescence filters are available on this system (excitation, emission):

  • 340-390, >410 (e.g. DAPI, Hoechst)
  • 455-495, >510 (e.g. FITC, GFPs, Alexa 488)
  • 503-548, >565 (e.g. Cy3, rhodamine, RFPs, Alexa 546)
  • 570-630, >655 (e.g. Cy5, Alexa 633, Alexa 647)

For additional information and current protocols, visit the ThermoFisher website.

Consumables for laser capture microdissection are available at the ALMC

The following items are in stock:

CapSure Macro LCM Caps LCM0211 48 caps
PEN Membrane Glass Slides LCM0522 50 slides


Please call us at 882-4895 if you need additional Arcturus® supplies or reagents for LCM.

Protocols from Molecular Devices

Application Notes from Molecular Devices

 

Image analysis/processing

 

Sample preparation

 

Life Technologies Supply Center

ALMC has an on-site Life Technologies Supply Center stocked with fluorescent secondary antibodies and markers. Consumables for laser capture microdissection are also available. In addition to our normal inventory, clients may special order anything from the Life Technologies catalog without shipping fees.

File transfer system

ALMC has a server that can be remotely accessed from any computer on the MU campus. It serves as a TEMPORARY holding place for files to facilitate transfer of the data you generate at the core to a local computer in your lab/office. Please note that due to space limitations the storage space is cleared every morning at 8:00 am, such that your files will only be stored for a maximum of 24 hours and you need to transfer them to your computer on the same day they were placed on the server. For instructions on connecting to this utility contact ALMC staff.