Image cytometry goes multiphoton

IT would be impossible to pinpoint the exact moment when image cytometry was born. However, the origin of the first quantitative imaging instruments can be traced back to the early days of microspectrometry pioneered by Caspersson (1,2). By coupling Caspersson’s microspectrophotometer with a microscope and moving the specimen in x and y directions, it was possible to perform a raster scan and record the information about the absorption of each pixel (3). Interestingly, the origins of confocal optics also go back to a microscopic spectrophotometer, an instrument built by Hiroto Naora in Japan over 50 years ago (4). Although numerous impressive (and exotic by today’s standards) instruments were constructed, the development of automated image cytometry was quite slow until substantial progress in microelectronics allowed access to inexpensive computing power (5–8). Development in electronics and informatics and miniaturization of computers revived interest in image-based microscopy analyzers among the early pioneers in the field. In the early 1990s, Kamentsky reported the first of his highly successful new designs, dubbed LSC (laser scanning cytometer). The system used galvanometer-driven mirror, motorized stage, and PMTs for two-color analysis. Initial results showed great reproducibility and were comparable to ones obtained by flow cytometry, which by that time had become the gold standard for single-cell analysis. The LSC had a fundamental advantage over flow-based systems: it was able to relate immunofluorescence data to morphological information about every measured cell. Flow-based systems are very limited in this regard, offering only forwardand side-scatter detection as parameters that might be related to morphology (9). Like any great idea, modern image cytometry has been invented more than once, the second time under the name of ‘‘high-content screening’’ (HCS). Conceptually and instrumentally, HCS technology is very similar and often identical to modern image cytometry techniques. However, historically the focus of these two methodologies was not identical. Modern image cytometry evolved from early automated cytological imagers and incorporated the flow cytometry approach to data representation and analysis, whereas HCS was born as a cell-based extension of high-throughput screening (HTS) techniques used in drug development (10–12). However, as with image cytometry devices such as the LSC, the most important element of HCS systems is the informatics component, allowing rapid statistical analysis, data visualization, and classification (13,14). Likewise, the development of 3D microscopy was stalled for many years after the early prototypes had been built. The first US patent for the idea of confocal microscopy had been awarded to Marvin Minsky in 1961 (filed in 1957) (15). Six years later, the first purely analog mechanical confocal microscope was reported by Mojmir Petran and Maurice D. Egger (16). But it took another 20 years for the first commercial confocal microscope, the Bio-Rad MRC500 based on the design by Brad Amos, to appear (17). The road from theoretical analysis to practical application was even longer for imaging methods based on nonlinear optical phenomena, such as multiphoton microscopy. The theory of two-photon absorption and emission was developed by Maria G€ oppert-Mayer in her doctoral thesis prepared at the University of G€ ottingen (18). In the late 1970s Sheppard, Kompfner, Gannaway, Choudhury, and Wilson, working at the Oxford University proposed the use of nonlinear optical interactions in high-resolution laser scanning microscopy (19). This idea, expanded and implemented in practice by Denk et al. at Cornell, was reported in a famous 1990 Science paper (20). Soon, the US patent awarded to the Cornell group was licensed to Bio-Rad and two-photon microscopy instruments became commercially available.