Microscopy of Living Cells

Nonlinear Optical Microscopy

Three important goals of optical microscopy have traditionally been imaging with high lateral spatial resolution, 3-D sectioning capability and high contrast in order to characterize specimens. This drive has led to the evolution of many different types of linear microscopy. Fluorescence confocal microscopy achieves these three goals; however, it often requires the addition of exogenous fluorophores for imaging biological systems. Some dyes and fluorescent labels disturb the natural evolution of a biological system, adding ambiguity to observations made or killing the specimen outright. Also, using fluorescent microscopy, the fluorophores may bleach which can lead to cell damage. Raman microspectroscopy and infrared microscopy achieve contrast through probing the vibrational spectrum of the molecules present in order to achieve chemical selectivity. Unfortunately infrared microscopy achieves a poor resolution because of the long wavelengths used. Furthermore, water absorption is a problem in many biological samples As for Raman microspectroscopy, it normally exhibits a very weak signal (most molecules have a small Raman cross section) which necessitates long integration or high laser intensities. High intensities create a large background auto-fluorescence signal that reduces contrast as well as cause damage to tissue. These issues may be overcome using nonlinear optical processes in combination with a confocal microscope.

One of the key advantages of nonlinear optical microscopy is an enhanced spatial resolution. Nonlinear processes rely on high intensities in order to generate appreciable signal. These high intensities can be generated using tight focussing of the incoming light using high numerical aperture lenses. The effective excitation volume generating the nonlinear signal will be smaller than that of a linear signal because of the requirement for high intensity (smaller by a factor of for second order processes and for third order ones). Furthermore, since the signal is generated only from the focal volume, this provides a natural way to achieve three dimensional sectioning without the need for confocal geometry.

A second advantage of nonlinear optical microscopy is that the excitation wavelength can be longer than in linear microscopy. While making use of this somewhat offsets the advantage of the enhanced spatial resolution, it offers several distinct advantages if one is examining condensed media. By using near-infrared light, it is possible to reduce absorption of the excitation pulses in the solvent which allows imaging of specimens embedded in condensed matter. This property assists in imaging live cells in vivo and imaging tissue. The reduced absorption also reduces the energy deposited in the sample which reduces sample damage.

A third advantage is the ability to achieve chemical specificity without the addition of exogenous fluorophores. This is possible by making use of resonant energy levels in the molecules of interest to greatly enhance the desired output signal. By avoiding the use of fluorescent tags, this allows for the in vivo or in vitro study of live cells without disturbance of natural cellular processes.

A final advantage is the low average power necessary if one employs ultrafast laser pulses. The field of ultrafast lasers is now mature and devices are now available which are tunable over a wide frequency range, making it possible to generate large nonlinear signals while using a low average power. This eliminates the problem of cell damage that can occur when dealing with linear microscopy (and associated bleaching effects). Also, with less energy deposited into the sample, the overall system suffers less negative effects due to the excitation (e.g. heating).

Figure 1: NLO microscopy of tissue from a joint replacement patient to screen for shed matallic nanoparticles. Implants can shed metals and metal oxides, which often have strong NLO interactions and therefore show up as bright signals. The streaking to the right side of the bright spots is due to distcharge of PMT detector during left to right raster scanning. Image Field = 175 micrometers. [with Isabelle Catelas, University of Ottawa & Ottawa Hospital]