The current state of Confocal Scanning Laser Microscopy Hjalmar Brismar Cell Physics, KTH

What are we doing in Cell Physics Confocal microscopy –History –Present Applications Areas of development –Excitation –Detection –Scanning

Cell Physics Study the biological cell from a physical perspective –Use tools and concepts from physics on biological problems –Develop methods and techniques –Describe biological functions and systems within a physical/mathematical framework We focus on: –Cell volume Osmolyte transport Water transport –Cell mass Measurement techniques Cell cycle/cell mass regulation –Intracellular signalling Frequency modulated Ca2+ signals

Instrumentation Microscopy (widefield, confocal, multiphoton) –Fluorescencent probes –Fluorescent labels, antibodies –Genetically engineered, GFP Electrophysiology –Patch clamp –MEA, multi electrode arrays

Confocal microscopy Marvin Minsky, 1955 –Laser (1958)1960 –Affordable computers with memory > 64kB –CSLM

WidefieldConfocal

Confocal evolution 1 st generation CSLM (1987) –1 channel fluorescence detection –50 Hz line frequency 2nd generation (commercial systems ca1990) –2-3 channel detection –>=100 Hz 3rd generation (1996) –4 channel detection –500 Hz 4th generation (2001) –32 channels –2.6 kHz –AOM, AOBS control

Confocal industry Carl Zeiss (physiology, dynamic measurements) Leica (spectral sensitivity) Biorad (multiphoton) (olympus) (nikon) (EG&G Wallac) …

Zeiss 510

Spectra Physics Millenia X - Tsunami

Leica TCS SP

Spectra Physics 2017UV

Applications - Techniques GFP –FRAP –FRET Multiphoton excitation

GFP- Green Fluorescent Protein Aequoria Victoria

GFP Discovered 1962 as companion to aequorin Cloned 1992, expression Aminoacids kDa Fluorophore made by 3 aminoacids (65-67) ”protected” in a cylinder

Dynamics GFP-Tubulin in Drosophila

Protein mobility – bleaching experiments Bleach mobile immobile FRAP – Fluorescence recovery after photbleaching

Variants of FP –Blue BFP –Cyan CFP –Green GFP –Yellow YFP –Red DsRed HcRed GFP timer CFP YFPCFP GFP

Fluorescence Resonance Energy Transfer FRET Spectral overlap Distance <10 nm DonorAcceptor

Interaction - FRET ( Fluorescence Resonance Energy Transfer) ProteinA CFP ProteinB YFP < 5-10 nm Excitation nm Emission >570 nm Donor Acceptor

FRET: NKA – IP 3 R

NKA – IP 3 R Donor GFP-NKA Acceptor Cy3-IP 3 R Before After Donor diff Photobleaching of acceptor removes FRET detected as increased donor signal Distance < 12 nm Ouabain binding to NKA shortens the distance – stronger interaction – increased FRET efficiency 15-25%

FRET based Ca 2+ sensor YFP CFP CaM 440 nm 480 nm YFP CFP CaM 535 nm 440 nm + 4 Ca 2+

Multiphoton excitation 1-photon 2-photon

Builtin confocality 1-photon 2-photon

PMT KonfokalMultifoton

 m 1-photon2-photon Better penetration (2-400  m) Enables measurements from intact cells in a proper physiological environment. Electrophysiology

FRET CFP-YFP multiphoton 790 nm 514 nm CFP – YFP separated by a 6 aminoacid linker Fluorochrome distance 5 nm YFP – Calcyon No excitation at 790 nm YFP excited at 880 nm

Development - Excitation Currently used lasers –Ar ion, 458,488,514 nm –HeNe 543, 633 nm –Ar ion 351,364 nm –ArKr 488,568 nm –HeCd 442 nm –Diode 405 nm –HeNe 594 nm –Multiphoton excitation, TiSa We need affordable, low noise, low power consumption lasers nm !

Development - Detection Spectral separation –Optical filters –Prism or grating Detectors –PMT –Photon counting diodes We need higher sensitivity, QE !

Development - Scanning Speed Flexibility

Ultrafast 3D spline scan Biological motivation –Ca2+ signals Measurement approach –Intracellular ion measurements –Combined electrophysiology

Frequency modulated Ca2+ signals

Data from live cell experiments combined with biochemical data is used as input for mathematical modeling-simulations [Ca 2+ ] Ca - wave Models verified by experiments can provide new information and direct the further investigations

Approach High resolution 3D recording of Ca2+ High speed recording Combined CSLM - electrophysiology Big cells – hippocampal pyramidal neurons

Scan speed

Confocal - line scan High time resolution (ms) Scan geometry  cell geometry 2D – cell cultures 2 s.

Arbitrary scan – 2D (Patwardhan & Åslund 1994)

2D specimen

Tissue – 3D cells

3D arbitrary scan x y z

Design criteria Z-axis precision >= optical resolution Bidirectional scan (to gain speed) Focusing distance um >100 Hz Nonharmonic

Ideas for ultrafast 3D scan Stage scan –High mass, impossible patch clamp Scan objective –Well defined mass, side effects in specimen ? Scan focusing lens inside objective –Tricky optics ?

Piezo focus with specimen protection 40X/0.9NA V/I