Leica_Mark_&_Find

23.09.2005

10:00 Uhr

Seite 6

Mark & Findand Tile Scan
Motorized Stage Applications
LAS AF

Leica_Mark_&_Find

23.09.2005

10:00 Uhr

Seite 1Motorized Stage Applications –Mark & Find and Tile Scan
Mark & Find and Tile Scan are part of the basic Leica Application Suite
for Advanced Fluorescence. When using a motorized stage, both modules are activated to give full advantage of a high precision stage over amanual stage. Mark & Find enables the user to define multi position
experiments to observe a large number of cells within one experiment,saving precious time. The Tile Scan feature allows the acquisition ofoverview images of the specimen with multiple channels and in threedimensions.
Mark & Find and Tile Scan are available on both the Leica AF6000
system series for widefield imaging and analysis and the Leica TCS SP5
confocal systems.

Mark & Find
High precision positioning is even more important when working withlive cells. Preparing a live cell experiment not only takes time but also
occupies the resources of the laboratory such as incubators, cleanbenches, etc. The experiment itself might run over hours or even severaldays. During the course of the experiment the acquisition system is outof use for other experiments. In the worst case scenario, the cells underobservation do not show the desired behavior or do not react to thereagents added during the course of the experiment and the experimenthas to be repeated. Precious time and resources are lost.
Such situations can be avoided with the Leica Mark & Find featurein combination with a high precision stage. These stages allow very
precise repositioning of previously defined locations. Multiple positions, for example multiple cells in the same Petri dish, can be storedin advance of performing the experiment.

Fig. 1: Mark & Find interface: Perform multi positioning
experiments and define location specific stack sizes.

Leica_Mark_&_Find

23.09.2005

10:00 Uhr

Seite 2

How it works
With the Mark & Find tool the user can define the current position of thesample and recall it later on from a list together with other previously
stored positions. This enables the user to quickly step through several
positions to compare them or record multiple time series in a pseudoparallel manner.
The user observes the live image and moves the stage to an interestingcell or location. Once the mark button is clicked, the x, y and z values ofthis position is stored. This procedure can be repeated for an unlimited
number of additional positions. At each stage position a cell specific z
stack size can be defined. Mark & Find can be combined with different
contrasting methods such as fluorescence and DIC and with otherexperimental set-ups, for example, time-lapse.
Powerful in live cell imaging
A simple, yet powerful application in live cell imaging is to mark miscellaneous positions of positive transfectants while searching for the mostappropriate location. This helps users in the time-consuming processof screening through their sample, as they can always go back to aprevious location if it is the right one.
Record multiple time series simultaneouslyMark & Find is particularly useful for studying dynamic processes inlive cells. In many cases one needs to block and induce the biologicalprocess of interest by adding and removing drugs or replacing incubation media. The difficulty is then to quickly find a typical cell whichdisplays the expected behavior during the course of the experiment.

Fig. 2: Capture all cell dynamics by recording multiple image series
in a single experiment.
A) Three positions have been marked prior to initiation of theexperiment.
B) Multiple movies can be recorded in a quasi-parallel mannerand compared in real-time or stored for off-line quantification.

A – Acquisition

B – Image series

yPos. 1áPos. 2

Pos. 3xt= 0123

Leica_Mark_&_Find

23.09.2005

10:00 UhrSeite 3In such a case, Mark & Find is very useful, because the user is ableto mark multiple cells previous to induction (Fig.2 A). By recording atime series in conjunction with Mark & Find, it is now possible to record
multiple cells in a quasi-parallel manner (Fig. 2 B). The chances ofobserving a process of interest in one single experiment are much higher
compared to the conventional situation where only one cell can beobserved at a time.
In effect, Mark & Find can help the user to save time or observe a larger
number of cells. If the aim is to obtain quantitative parameters fromthe experiment (i.e. size, migration speed, particle tracking) a gain inthroughput will effectively improve the statistical significance of thedata.
Mark & Find can be applied over several rounds of cell cycles, too.
Additionally, multiple cells can be observed and their behavior can becompared.
Applications in developmental biology
Since Mark & Find can be combined with both t-series and z-scans,very complex recording scenarios become possible such as following
dynamic processes in large specimen at multiple positions over time,each with an independent z-stack. Applications in developmental biology are perfectly suited to such tools, for example to follow the development of the nervous system and transport systems of animal models.
Figure 3 shows a real-world example of a time-lapse recorded using
Mark & Find. NIH 3T3 mouse fibroblast cells expressing H2A-YFP stably
and were incubated with Aphidicolin over night. This leads to an arrestin the G1-/S-phase transition. The shown time series was observed 4.5
hours after removal of the blocking agent. The cells were monitored foranother 4 hours. In total 10 positions were selected using Mark & Find
over time, and z-stacks of 7-8 µm with a z-step size of 0.3 µm wererecorded for each cell. Out of those ten cells, only one could be followed
through mitosis (left column), whilst the others did not divide during theobservation time (right column). This is a typical situation in live cell
imaging when biological processes are triggered using specific drugs.
Mark & Find increases the chances of observing such a process in asingle experiment. DIC (differential interference contrast) and fluorescence images of the cells are shown over time from the top to the bottom. All DIC images are single in focus slices of their respective z-stacks,
H2A-YFP images shown as maximum projections over the whole stackafter deconvolution. Magnification 63x objective.

mitosis observed
DIC
H2A-YFPno mitosis observed
DIC
H2A-YFP

t1

t2

t3

t4

t5

t6

t7

Fig 3: NIH 3T3 mouse fibroblast cells expressing H2A-YFP.
Two positions of a Mark & Find experiment.
Courtesy of Constantin Kappel, DKFZ, Heidelberg, Germany

Leica_Mark_&_Find

23.09.2005

10:00 Uhr

Seite 4Tile ScanThe Tile Scan module allows the user to automatically assemble animage from multiple smaller images. Large specimens that are bigger
than the current field of view can be viewed as a whole using the Tile
Scan function. This works by taking a number of adjoining images of thesample (the “tiles”) and merging them digitally to a larger view of thescene.
How it works
A tile scan area can be defined by marking just two opposite positions
defining the outer points of the area to be scanned. The positions aremarked in the same way as locations in Mark & Find. Depending on thefield of view, the Tile Scan feature will calculate the number of tiles
needed to cover the scan field.
Produce high resolution overview images
Figure 5 shows reconstructed Drosophila larval salivary glands takenwith a 20x objective lens. The user is presented with the option of preserving the original images as well as the overview image for reference.
Since the automated stage under full software control, the user only
needs to specify the boundaries of the area of interest, the software
will automatically calculate the area to be scanned.

Fig. 4: Tile Scan interface: Get an overview image ofyour specimen with multiple channels and combine
it with z-stacks.

Find live transfectants fast
In the live cell imaging of transiently transfected cells, one often faces
the problem of finding a cell expressing the protein of interest at thecorrect level. With Tile Scan it is now possible to produce a “bird’s eyeview” image of the entire coverslip or culture dish to aid the user infinding interesting parts of the sample more quickly. Such an overview
image can serve as a reference for the whole experiment and can also
be used to obtain important quality parameters, for instance cell density,transfection efficiency or cell viability. An added advantage is that thecell, which was captured in more detail, can still be viewed in its context
later on.

Author: Constantin Kappel, Div. Theoretical Bioinformatics,
German Cancer Research Center (DKFZ), Heidelberg, Germany
in cooperation with Leica Microsystems

BFig. 5: Drosophila salivary glands at 20x magnification with bright fieldand fluorescence channels overlaid. The whole image was assembled
from 5 x 3 single images at full resolution (1392 x1040 per image). Image
shows nuclei (DAPI) in blue, Neurons/Axons (Alexa 488/Cy3) in green
and neuronal nuclei (Alexa 594) in red. Note; some crosstalk between
Cy3 and Alexa 594 is visible, as the image was captured with a broadband pass BGR cube. The merged image is completely unprocessed.
Courtesy of Dr. Christoph Melcher, Research Center Karlsruhe,Karlsruhe, Germany©

Copyright Leica Microsystems CMS GmbH • Am Friedensplatz 3 • D-68165 Mannheim, Germany • Tel. +49 621/70 28 0 • Fax +49 621/70 28 10 28 LEICA and the Leica Logo are registered trademarks of Leica IR GmbH.
Order no: 15915321 • September 2005

Leica_Mark_&_Find@23.09.2005
10:00 UhrSeite 5

www.confocal-microscopy.com