Light Exposure in Microscopy How can Cell Survival be Increased? - - PowerPoint PPT Presentation

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Light Exposure in Microscopy How can Cell Survival be Increased? - - PowerPoint PPT Presentation

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Light Exposure in Microscopy How can Cell Survival be Increased? Herbert Schneckenburger, Sarah Schickinger, Petra Weber, Michael Wagner, and Thomas Bruns Aalen University, Institute of Applied Research, 73428 Aalen, Germany Fluorescence

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SLIDE 1

Light Exposure in Microscopy – How can Cell Survival be Increased?

Herbert Schneckenburger, Sarah Schickinger, Petra Weber, Michael Wagner, and Thomas Bruns Aalen University, Institute of Applied Research, 73428 Aalen, Germany

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SLIDE 2
  • How much light do we need for

microscopy and how much light can we apply to living cells?

  • Can we use or even exceed solar

irradiance?

  • How long will cells endure this

irradiation? Solar irradiance: 1 kW/m² = 100 mW/cm² = 1 mW/mm² = 1 nW/µm² 1 J/cm² = 10 s of solar irradiation

Fluorescence Microscopy of Living Cells

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SLIDE 3

Viability of U373-MG Glioblastoma Cells upon Irradiation

(colony formation assay; native cells)

4 min. 16 min. 32 min. solar irradiaton

7 days Surviving cells

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SLIDE 4

Cell Viability upon Irradiation

  • native cells and fluorescent markers -

Cell line Marker Conc. [µM] ex [nm]

  • Max. light

dose [J/cm²] Solar exposure time [s] U373-MG  375 25 250 U373-MG  514 100 1000 U373-MG  633 200 2000 U373-MG Laurdan 8 391 10 100 CHO-K1 DiA 5 488 10 100 CHO-K1 DiO 5 488 10 100 CHO-K1 GFP-Mem 488 10 100 CHO-K1 R 123 5 488 520 50200 CHO-K1 MTO 0.05 514 50 500 CHO-K1 GFP-Mito 488 5 50

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SLIDE 5

Prior tp illumination After illuminaition

2.6 J/cm2 3.9 J/cm2 55.4 J/cm2

Example: 3T3 Fibroblasts + Acridine orange (5 µM, 30 min.)

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SLIDE 6

Fluorescence Microscopy with Axial Resolution

Methods:

  • Laser Scanning

Microscopy (LSM)

z = 10 µm z = 15 µm z = 60 µm

  • Structured

Illumination Microscopy (SIM)

  • Light Sheet

Only planes under

Fluorescence

investigation are illuminated

Microscopy (LSFM)

 minimum light exposure

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SLIDE 7

Maximum Light Exposure to Living Cells in 3D Microscopy

Method Experiment Max.light dose [nJ/µm²] Irradiance [nW/µm2]

  • Max. no. of

images Widefield microscopy Autofluor. 250 1 250 LSM

  • Fluor. marker

100 1 20 Light Sheet (N layers)

  • Fluor. marker

100 1 N  100 TIRFM

  • Fluor. marker

300 1 100300 Single Molecule Methods

  • Fluor. Marker

(low conc.) 2,000 500  1 STED (650 nm)

  • Fluor. Marker

100500 30,000 not relevant

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SLIDE 8

Total Internal Reflection Fluorescence Microscopy (TIRFM)

Membrane Associated Paxilline (Pax-EYFP) / Focal Adhesions

Conventional fluorescence microscopy TIRFM ex = 470 nm; d  530 nm

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SLIDE 9

Variable-angle Total Internal Reflection Fluorescence Microscopy (TIRFM)

monomode fiber microscope

  • bjective

lens hemi-spherical prism concave mirror adjustable mirror step motor light trap deflection mirrors

 (nm)

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SLIDE 10

Nanometre Cell-Substrate Topology of Glioblastoma Cells

  • using the fluorescent membrane marker laurdan -

U251-MG tumour cells U251-MG with tumour suppressor gene TP53 Cell-substrate topology offers a criterion to distinguish tumour cells and less malignant cells

Cells provided by J. Mollenhauer, Dept. of Molecular Oncology, University of South Denmark, Odense

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SLIDE 11

Light Sheet Fluorescence Microscopy (LSFM)

telescope cylindric lens deflection mirror adjustable screw

  • bjective turret

micro-capillary microscope

  • bjective lens

light trap laser excitation

  • T. Bruns, S. Schickinger, R. Wittig and H. Schneckenburger, "Preparation strategy and illumination of 3D cell cultures

in light sheet-based fluorescence microscopy," J. Biomed. Opt. 17, 101518 (2012).

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SLIDE 12

Light Sheet Fluorescence Microscopy (LSFM)

(CHO-GFP-Mem)

Selected parameters: Beam waist: z = 510 µm Beam width: y  8 mm Focal depth: x  150200 µm

200 µm z = 40 µm z = 80 µm 3D

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SLIDE 13

LSFM Application: 3D Imaging of Necrotic Cells

(Rotenone: 1 µM, 3 h; CellTox: 2 h; ex = 470 nm, d  515 nm)

Single Plane: z = 50 µm; d  10 µm 3D Reconstruction

100 µm

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SLIDE 14

LSFM Application: Uptake / Interaction of a Cytostatic Drug

MCF-7 Breast Cancer Cells, Doxorubicin: 8 µM, 6 h, ex = 470 nm, d  515 nm Transillumination Fluorescence (single plane) Fluorescence Lifetime Fluorescence lifetime imaging (FLIM) is used to probe intermolecular Interactions of doxorubicin and to identify a degradation product

100 µm

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SLIDE 15

Förster Energy Transfer (FRET) Based Sensor for Apoptosis

Non-radiative energy transfer from enhanced cyan fluorescent protein to enhanced yellow fluorescent protein via a cleavable peptide linker DEVD

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SLIDE 16

Fluorescence Spectra prior to and subsequent to Apoptosis

ex = 391 nm (ECFP)

ECFP EYFP

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SLIDE 17

Fluorescence Decay Profiles prior to and subsequent to Apoptosis

  • HeLA-Mem-ECFP-DEVD-EYFP -

0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1 1000 2000 3000 4000 5000 6000

Time [ps] Relative Fluorescence Intensity Control Staurosporine (2 µM, 2.5 h)

Sampling gate

I = I0 e -t/

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SLIDE 18

FRET-Based Membrane Assiciated Sensor for Apoptosis

  • LSFM / FLIM of ECFP in HeLa Cells; λex = 391 nm -
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SLIDE 19

10 s 30 s 90 s 100 µm Flow direction Illumination

0.5 1 1.5 2 2.5

Ratio I391/I470

÷ =

λexc = 391 nm λexc = 470 nm Ratio I391 / I470

LSFM Application: Redox Imaging upon Addition of H2O2

U251-MG glioblastoma cells with redox sensitive Grx1-roGFP2

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SLIDE 20

Summary

  • Light exposure in microscopy is limited due to phototoxicity
  • Maximum light doses for cell survival - dependent on wavelengths

and fluorescence markers – are typically in the range of 5200 J/cm² corresponding to 50 s  2000 s of solar irradiance

  • Light sheet fluorescence microscopy (LSFM) needs minimum light

doses for 3d samples, e. g. multicellular tumour spheroids (examples including apoptosis and necrosis, uptake of cytostatic drugs, redox imaging)

  • TIRFM needs minimum light doses for cell surfaces or membranes

(examples including focal adhesions, cell-substrate topology)

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SLIDE 21

Acknowledgment / Literature

Projects are funded by Land Baden-Württemberg, the European Union (Europ. Fonds für die Regionale Entwicklung) as well as Bundesministerium für Wirtschaft und Energie (ZIM, grant no. KF 2888104UW3). The authors thank B. Angres and NMI Reutlingen for providing HeLa cells expressing the FRET sensor as well as R. Wittig (ILM Ulm) for his cooperation.

  • H. Schneckenburger, P. Weber, M. Wagner, S. Schickinger, V. Richter, T. Bruns, W.S.L. Strauss, R. Wittig: Light

exposure and cell viability in fluorescence microscopy, J. Microsc. 245 (2012) 311318.

  • M. Wagner, P. Weber, H. Baumann, H. Schneckenburger: Nanotopology of cell adhesion upon variable-angle total

internal reflection fluorescence microscopy (VA-TIRFM), J. Vis. Exp. 68 (2012) e4133.

  • T. Bruns, S. Schickinger, H. Schneckenburger: Single plane illumination module and micro-capillary approach for a

wide-field microscope, J. Vis. Exp. 15(90) (2014) e51993.

  • S. Schickinger, T. Bruns, R. Wittig, P. Weber, M. Wagner, H. Schneckenburger: Nanosecond ratio imaging of redox

states in tumour cell spheroids using light sheet based fluorescence microscopy, J. Biomed. Opt. 18(12) (2013) 126007.

  • P. Weber, S. Schickinger, M. Wagner, B. Angres, T. Bruns, H. Schneckenburger: “Monitoring of apoptosis

in 3d cell cultures by FRET and light sheet fluorescence microscopy”, Int. J. Mol. Sci. 16(3) (2015) 53755385.