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Equipment

Equipment

Transmission Electron Microscopy

Transmission electron microscopy (TEM)  allows visualisation of morphology and structural details of biological and material samples such as: organelles, proteins, cells membranes, polymers, vesicles, exosomes, nanoparticles, semiconductors, crystalline structure. TEM uses a transmitted electron beam that passes through the specimen to form an image. The specimen must be less than 100 nm thick and specially prepared. The resolution that can be reach with this microscope is around 0.3nm.

Girl analysing micropscope images at computer in lab

Instruments:

Joel JEM -1400 Plus Transmission Electron Microscope

  •  Acceleration voltage 40kV to 120kV
  •  Magnification x10 to x800,000
  •  JEOL Ruby 8MP Bottom mounted CCD digital Camera
  •  Standard specimen holder
  •  +/- 70º tilt with support for tomography
  •  High Tilt tomography holder
  •  Liquid Nitrogen Cold Trap -Cryo ready
  •  2D montaging and 3D tomography
  •  Selected area electron diffraction

Related links

Jeol UK

Jeol Europe

MyScope Australia

Products - Transmission Electron Microscope (TEM)

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Automated  tissue processor Leica EM-TP  

Allows automatic biological sample preparation for transmission electron microscopy.  It has a heating/cooling system that maintain constant processing temperature for over 24h. The carousel holds 24 vials and utilizes specialized baskets and capsules that allows exchange of buffers. An exhaust system supports safer use of toxic substances.

Related Links: 

Leica Microsystems

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LEICA Ultracut - UCT ultra-microtome

Uses a glass or diamond knife to cut semi and ultrathin sections to prepare electron microscopy samples. Sections can be between 70-100 nm thick and 0.5 to 2mm wide and can be stained and mounted on the TEM grids for visualization.

Related Links: 

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Widefield fluorescence microscopy

Widefield fluorescence microscopy is an optical technique where the sample is illuminated with visible light of a specific wavelength to excite fluorophores attached to different molecules in cultured cells or tissues. This technique can be used to identify cells, cell membranes, organelles, proteins, lipids in live or fixed samples.

Girl using equipment

INSTRUMENTS

Leica DM5500B  Upright Microscope

Can be used for fixed samples only. 

Allow examination of histopathological  samples with Leica  DFC 290 colour digital camera.

Bright field  and fluorescence images  can be recorded using  Leica DFC340 monochrome  digital camera.  

Objectives :  

  • 2.5x/  0.07 NA PL FLUOTAR
  • 5x/     0.15 NA HCX PL FLUOTAR
  • 10x/   0.3 NA HCPL FLUOTAR Ph1
  • 20x/   0.5 NA PL FLUOTAR-Ph2
  • 40x/   0.85 NA HCX PL APO, CORR
  • 100x/ 1.4-0.7NA HCX PLAN APO oil 

Filter cubes

1.Excitation: 360nm±40;Emission: 470nm±40

2.Excitation: 480nm±40;Emission: 527nm±30

3.Excitation: 560nm±40;Emission:645nm±75

4.Excitation: 620nm±60;Emission:700nm±75

Related Links: 

More information

Leica LASX Acquisition software 

LASX software 

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Nikon AZ100 Multi Zoom upright microscope

It is a compound microscope designed to visualize  large specimens in epifluorescence or DIC (Differential Interference Contrast) methods.  With  low magnifying, long working distance objectives and a zooming mechanisms  it  digitally magnify  between  5x to 400x.  

Histopathological  samples can be visulaized too due to the color camera.

Objectives:

  • AZ Plan Apo   1x NA 0.1 (working distance 35mm)
  • AZ Plan Fluor 2x NA 0.2 (working distance 45mm)
  • AZ Plan Fluor 5x NA 0.5 (working distance 15mm)

By an optical zoom mechanism each objective's magnification can be increased up to x 8 resulting in 40x (with objective 5x and zoom-factor 8x) magnification.

Filter cubes:

1.Excitation:340nm-380nm;Emission:435nm-485 nm

2.Excitation:465nm-495nm;Emission:515nm-555 nm

3.Excitation:540nm±25nm ;Emission:605nm±55nm

Camera:

Nikon DS-Ri1 colour camera. Two resolution settings are possible: 1636 x 1088 pixel or 4908 x 3264 pixel.

Related Links: 

Nikon Multizoom website

Nikon Multizoom movie

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Nikon 6D Live Cell Imaging inverted Microscope

It is an automatic system that allow multi-dimensional time-lapse imaging  of live or fixed cultured cells in multi-well plates, slide or petri dish  within a controlled environment. Tile scanning on the fixed or live samples is possible.

Perfect Focus System (PFS)  corrects focus drift in real time during the multi point acquisition.

Objectives:

  •  10x /   0.13  NA CFI60 Plan Fluor DLL
  •  20x /   0.75  NA CFI60 Plan Apochromat VC
  •  40x /   0.6    NA CFI60 Super Plan Fluor ELWD
  •  40x /   1.3    NA CFI60 Plan FLUOR oil
  •  60x /   1.4    NA CFI60 Apochromat Lambda S oil
  • 100x/   1.45  NA CFI60 Plan Apochromat Lambda oil

 

Filter cubes:

1.  Excitation: 405 nm; 470 nm; 630nm

2. Excitation: 385 nm; 505 nm; 525 nm; 560 nm; 625nm

3. FITC

4. Texas Red

Camera:

Neo 5.5 sCMOS ANDOR Camera with 5.5Megapixels and 2560x2160 16.6 mm x 14.0 mm sensor size.

Light source:

Lumencor  Spectra X- LED Light Engine with six solid-state light sources that generate seven colour bands (violet, blue, cyan, teal, green, yellow and red).

Related Links: 

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EVOS- FL AUTO 2 CELL IMAGING SYSTEM

EVOS is an automated inverted four-channel fluorescence and transmitted light imaging system  that can be used for imaging fixed or live biological samples in well plate or fluorescence slides. The colour camera allows imaging of  fixed histopathology samples.

Time lapse images can be recorded for live cells with the environmental control.

EVOS FL Auto 2 system can be programmed to run well-plate (plastic or glass) scanning, time lapse experiments, and tile-stitch/montage area scans—in Z-stack and/or time-lapse modes.

Objectives:

  •  4x /    0.13 NA  AMG LPlan FL PH
  • 10x/    0.25 NA  AMG LPlan FL PH
  • 20x/    0.4    NA  AMG LPlan FL PH
  • 40x/    0.65  NA  AMG LPlan FL PH
  • 40x /   0.75  NA  Plan FL Cover Slip

Filter cubes:

1.  DAPI:Excitation: 360nm; Emission: 447 nm

2.  GFP : Excitation: 470 nm;Emission: 535nm

3. Texas Red: Excitation: 530 nm;Emission: 593nm

4.  CY5:          Excitation: 628 nm;Emission: 692nm

 

Camera: 

-1.3 MP CMOS monochrome camera with 1328x1048 pixels

-High sensitivity 3.2 MP CMOS color camera with 2080x1552 pixels

Related Links: 

Thermo Scientific

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Confocal Microscopy

Confocal microscopy is an optical technique that eliminates out of focus light by adding a pinhole in the light path. Laser scanning confocal microscopy produces thin (0.1 to 1.5 micrometre) optical sections through fluorescent specimens that have a thickness of 100 micrometres or more.

Confocal microscopes can be used to visualize the structure of cells, drug distribution in the cells, organelles, cell membranes protein distribution or interactions. Using the FRET (Forster Resonance Energy Transfer) or FRAP (Fluorescence Recovery After Photo bleaching) module it is possible to detect if two molecules are within 10 nm of each other or to investigate the diffusion or movement of the molecules in a specific area of the cells or tissue.

Student using Advanced Imaging microscope

Instruments

Leica TCS-SP5 inverted microscope

This can be used for fixed or live samples. It uses a spectral detection, that mean all dyes that can be excited with the availble lasers can be detected.

Lasers

  • 405 nm Blue diode laser
  • Argon laser with excitation wavelengths 458 nm, 476 nm, 488 nm, 496 nm and 514 nm
  • 561 nm DPSS-Diode
  • 594 nm Helium Neon Gas laser
  • 633 nm Helium Neon Gas laser

Objectives:

  •  20x /   0.5NA  HC PL Fluotar
  •  20x /   0.7NA  HCXPL APO Lambda blue IMM UV
  •  40x /   0.75 NA HCXPL Fluotar
  •  40x /   1.25 NA HCX PL APO, PH3 -oli
  •  63x /   1.4 NA HCX PLAPO Lambda blue- oil
  • 100x/   1.4NA HCX PL APO -oil

 

Software for image acqusition : LAS AF 

LAS User interface movie

Leica confocal microscopy webinar

Leica SP5 introduction

Leica software

Related Links: 

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Leica TCS SP8  inverted microscope

Leica TCS SP8 confocal has been designed with optimal photon efficiency and high speed.  This system has 4 detectors (2 sensitive Hybrid GaAsP and 2 PMT) and a motorized xyz-stage.

Time lapse imaging of live cultured cells or tissue is possible due to the controlled environment.

Because of the spectral detection any dyes that can be excited with the avaiable lasers can be detected.

Resonant scanner for fast image aqusision is available.

Objectives:

  •  10x /   0.4   NA   HC PL FLUOTAR
  •  20x /   0.75 NA   HC PL APO CS2
  •  40x /   1.10 NA   HC PL APO W CORR CS2 water
  •  40x/    1.25 NA   HCX PL APO PH3 oil
  •  63x /   1.4   NA   HCX PL APO lambda blue oil
  • 100x/   1.4   NA   HC PL APO CS2 oil

Laser lines for excitation:

  • 405 nm
  • 488nm
  • 510 nm
  • 552nm
  • 638nm

Related Links: 

LAS X software for  image processing

LAS X image acqusition movie 

Spectral detection with Leica SP8

Automated imaging with Navigator

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Nikon C1 inverted microscope

Lasers:

  • 405 nm diode laser
  • 488 nm diode laser
  • 561 nm diode laser

 

Objective:

  • 10x /  0.3NA Plan Fluor
  • 20x /  0.45 NA Plan Fluor ELWD
  • 40x /  0.75 NA Plan Fluor
  • 40x /  0.6NA SPlan Fluor 
  • 60x / 1.00 NA water 

 

Related Links: 

Software for image acqusition:  EZ-C1 3.6

Nikon C1

Nikon Ti Eclipse

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Leica SP8- upright microscope

This microscope is designed mainly for living samples. It can be used for fixed samples too and immunofluorescence detection.

Spectral detection via 2 PMT’s and 2 sensitive Leica Hybrid detectors.

Resonanat scanner for fast image acqusition is available.

Objectives

  •  10x /  0.4NA    HC PL APO, PH1
  •  10x /  0.3NA    HCX APO L W. water immersion
  •  20x /  0.5NA    HC PL FLUOTAR
  •  25x /  0.95NA  HC FLUOTAR W VISIR; High transmission >83% from 400nm to 1300nm. Colour corrected for VIS and NIR up to 950nm.  water immersion
  •  40x /  0.6 NA   HCX PL FL L CORR PH2 02/C
  •  40x /  0.85NA  HCX PL APO CORR CS 0.11 superior colour correction
  •  63x /  1.2 NA   HC PL APO W CORR CS2; water immersion 
  •  63x /  0.9 NA   HC APO L W UV1 CS2 water immersion

Lasers

  • 405 nm diode laser
  • 488 nm diode laser
  • 514 nm diode laser
  • 552 nm diode laser
  • 638 nm diode laser

Related Links: 

Leica SP8 description

LAS X software for image acqusition and analysis

Leica confocal general theory 

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Leica TIRF - Total Internal reflection fluorescence inverted microscope

Many cellular events like viral infection, endocytosis, receptor-ligand interaction, cytoskeletal dynamics, movement of organelles in the vicinity of the membranes,  take place at the cell surface; with TIRF  those  events can be visualized  with low noise  or interfernce from near regions. Changes in the orientaion of the molecules can be detected with the polarization controled electromagneric field orientation.

Total internal reflection fluorescence (TIRF) or evanescent wave microscopy is a technique where fluorophores are illuminted by an electromagnetic field  that is generated by the total internal reflection of the  incident laser beam  at the interface of  media with different index of refraction like water and glass. Electromagnetic field restricted to 100nm-150nm deep into the specimen can excite  individual molecules with low background fluorescence , low light exposure and no out of focus fluorescence.  

 

 

800 533

Leica TIRF microscope uses objective to generate the evanescent electromagnetic field. 

Excitation lasers:

  • 405 nm
  • 488 nm
  • 561nm
  • 632 nm

Objectives:

  •  10x /   0.4NA    HC PL APO 
  •  20x /   0.7NA    HC PL APO 
  •  40X/    0.85NA  HC PL APO
  •  63x /   1.47NA  HC PL APO Optimized for TIRF oil immersion
  • 100x /  1.47NA  HC PL APO Optomized for TIRF oil immersion

Filter cubes:

1.QUAD cube for TIRF fast acqusition:

     Excitation : 405±10nm ; 488±13 nm; 561±10nm; 635±15nm

     Emission  : 450 ± 55nm ; 525±50nm ; 605±45nm; 730±100nm

2. CFP  Excitation  : 436±20nm ;  Emission  480±40nm

3. GFP  Excitation  : 470±40nm ; Emission  500±40nm

4. CY3  Excitation  : 545±25nm ;  Emission  595±50nm

5. CY5  Excitation  : 640±30nm ; Emission  690±50nm

 

Camera: 

-Andor Zyla 4.2 sCMOS Camera with 4.2 Megapixel, 82% QE

 

Links: 

-TIRF theory

 

Leica TCS SP8-MP - Multiphoton excited fluorescence upright microscope

Two-photon excitation microscopy (TPEM) is a nonlinear optical technique where fluorescent dyes are excited by absorbing the energy of two photons simultaneously at double the wavelength used in confocal microscopy. To excite fluorophores, multiphoton microscopy uses infrared pulsed laser beam that has lower energy than the visible light used in 1 photon microscopy.  Because of this, less light scatter so fluorophores in thicker tissue (1mm) can be detected and long-time imaging can be acquire because of less photo-bleaching and photo-destruction.

FLIM- Fluorescence lifetime imaging microscopy module is attached to the Leica TCS-MP microscope.

 

Excitation source:

 

Multi-photon laser: Mai Tai eHP DeepSee IR laser excitation from 690-1040nm, pre-chirped/short pulse width for compensation with deep tissue imaging- high performance / low scattering.

Spectra-Physics

Objectives:

  • 10x/ 0.4NA HC PL APO, PH1
  • 10x/0.3NA HCX APO L W
  • 20x/ 0.5NA HC PL FLUOTAR
  • 25x / 0.95 NA HC FLUOTAR L W VISIR , High transmission >83% from 400nm-1300nm. Colour corrected for VIS and NIR up to 950nm- water immersion
  • 40x /0.6NA HCX PL FL L CORR PH2
  • 40x / 0.85NA HCX PL APO CORR CS 0.11 superior colour correction
  • 63x / 1.2NA. HC PL APO W CORR CS2 water immersion
  • 63x/ 0.9NA HC APO L W UVI CS2 water immersion

 

 

Techniques

  • Fluorescence imaging
  • Spectral imaging
  • Fluorescence lifetime imaging with Pico Quant system 

Time Correlated Single Photon Counting (TCSPC) measures the fluorescence decay of the fluorophores. Fluorescence lifetime is an endogenous properties of the fluorophore defined as the average time that molecule remains in an excited state before deactivate and return to the ground state by emitting a photon. The image that creates it is based on the differences in the excited state decay rate (fluorescence lifetime) of the fluorophores.

Fluorescence lifetime does not depend on concentration, absorption by the sample, sample thickness, photo-bleaching and/or excitation intensity . At the same time, the fluorescence lifetime depends on a wealth of environmental parameters such as pH, ion or oxygen concentration, molecular binding or the proximity of energy acceptors making it the technique of choice for functional imaging of many kinds.

Related Links: 

Multi-photon theory

PicoQuant SymPhoTime64 software

Spectral imaging

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The system includes

  • Mai Tai eHP DeepSee IR laser tunable from 690nm to 1040 nm pre-chirped/short pulse width for compensation with deep tissue imaging- high performance / low scattering.
  • Resonant module scanner that allow acquisition of 30 fps @ 512x512.
  • 4 channel simultaneous acquisition using: 2 sensitive GaAsP, hybrid and two PMT detectors.
  • 2-Channel TCSPC Detector 
  • Automated stage for multi-position imaging
  • Incubation system for temperature, humidity and CO2 control

FLEXSTATION3 – Bench top multi-mode micro plate reader

The Molecular Devices FlexStation 3 is a benchtop scanning fluorometer and integrated fluid transfer workstation capable of conducting endpoint, kinetic, absorbion , emission spectrum scan,l uminescence and well scan experiments in a multi-well plate format (6, 12, 24, 48 and 96-well) and fluidics transfer experiments in 96-well format.  The FlexStation 3 is ideal for measuring the activity of receptors by  quantifying intracellular calcium release.  In addition,  the measurement of enzyme activity,  GMP and AMP levelspotassium ion channel flux, and membrane potential is possible. 

This multi-mode plate reader with 8 integrated liquid dispensers supports the following applications:

Automated system allowing rapid and high throughput screening of a large panel of biological parameters

  • Absorbance (200nm-1000nm)
  • Fluorescence (250 nm- 850 nm)
  • Luminescence (250-850 nm)
  • Dual monochromators (1nm)

Links:

 

 

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