MICRODISSECTION.pdf

8/11/2019 MICRODISSECTION.pdf

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a report by

D r Y i lma z N i y a z

Member, Gesellschaft fr Biochemie und Molekularbiologie (GBM) and Application Specialist for Biology,

P.A.L.M. Microlaser Technologies AG

Non-contact Laser Microdi s sec t ion and Pres sure Catapul t ing

A Ver sat i l e Too l for Spec i f i c Sample Generat ion

B U S I N E S S B R I E F I N G : F U T U R E D R U G D I S C O V E R Y 2 0 0 4

93

Screening & Image Analysis

I n t r o d u c t i o n

Modern molecular biomedical research relies on the

capability of pure sample preparation. Amongst various

methods for achieving homogeneous samples, only

laser microdissection and micro-manipulation offer

high-resolution control of sample composition byselecting or rejecting individual cells. This technique is

becoming more and more important for the

understanding of cellular physiology and pathology.

The combination of a highly precise microscope stage,

the RoboStage II, with a versatile robotic sample

collection device, the RoboMover, as realised in the

PALM MicroBeam HT laser microdissection system

(see Figure 1), enables high-throughput, non-contact

sample preparation. In principle, a pulsed nitrogen laser

is coupled through the epifluorescence path into an

inverted microscope and focused on a micron-sizedspot via the objective lenses. By this means, the

microscope known as an opto-analytical device turns

into a most versatile micro-manipulation tool

selected specimens of any origin can first be laser-

microdissected and, thereafter, ejected directly into a

capture device. The sample transfer is driven solely by

a laser-induced transportation process, the patented

Laser Pressure Catapulting (LPC) technology. Thus,

PALM MicroManipulating Systems have neither

physical nor mechanical contact with the specimen

and, as the extracted samples are traceably derived from

a defined origin, there is no risk of contamination forthe isolated samples. These samples can be applied to a

great variety of downstream applications, such as

(RT-) PCR analyses, microarray hybridisation of

cellular DNA and RNA, MALDI/SELDI mass

spectrometry or chromosomal analyses (see Table 1).

In addition, the same laser system can also be used to

microinject drugs or genetic material into living cells

without harming their viability, enabling genetic

engineering without mechanical tools, disturbing

chemical agents or viral vectors. With this unique

combination of microsurgery and non-contactsample preparation, laser microdissection and micro-

manipulation has become one of the foremost

emerging technologies for functional genomics and

proteomics; non-contact laser microdissection is

frequently performed in numerous research institutes

and industrial laboratories throughout the world.1

L a s e r M i c r o d i s s e c t i o n a n d L a s e r

P r e s s u r e C a t a p u l t i n g T e c h n o l o g y

Uniquely by the force of focused laser light, it ispossible to catch, sort and segregate, as well as to

microdissect and isolate, biological objects on a

microscopic scale. Therefore, lasers of high beam

quality are coupled into the path of a research

microscope and thereby focused through objectives

with high aperture to a diameter of less than one

micron. At this size, the user is able to manipulate at

the level of single cells or even, at an appropriate

magnification, at the level of sub-cellular

components. The pulsed (3ns) ultraviolet A (UVA)

laser beam used for LMPC impacts the sample at high

energy density (10MW/cm2

). This condensed UVradiation (l=337nm) within the focal spot is absorbed

by the sample. The energy transfer is sufficient to

break molecular bonds resulting in fragmentation of

the radiated matter, without any mechanical contact.

Thus, at the focal point, unwanted material is photo-

fragmented into small molecules and atoms, a

phenomenon that is called ablative photo-

decomposition or cold ablation. But, as this cutting

is a photochemical and fast process devoid of heat

transfer, adjacent biologic matter or biomolecules

such as DNA, RNA or proteins out of the focus are

not affected. Therefore, these molecules can beroutinely isolated from the specimen for downstream

analyses and applications; and even living cells can be

captured for subsequent cultivation.

After the cutting procedure, the selected area is

ejected from the object plane, usually with a single

laser shot. This LPC technology marks the

breakthrough in modern laser capture methods and

enables the entire non-contact preparation of pure

and homogeneous samples in a fast and elegant

manner. It is believed that the formation of a locally

restricted, expanding micro-plasma drives themicrodissected samples out of the plane. The

sample is transported with high speed (25m/sec)

over several millimetres against gravity, directly into

a capture device mounted within the laser beam

Dr Yilmaz Niyaz has been working

for P.A.L.M. Microlaser Technologies

AG as an application specialist for

biology since September 2003. He

wrote his doctoral thesis at the

University of Heidelberg at the

Institute for Molecular Genetics and

Evolution and the Institute for

Biological Chemistry at Prof. Dr. U.

Gehrings Laboratory (19992003).He began his studies in Biology at

the Freie Universitt Berlin and

completed his degree as molecular

biologist at the University of

Heidelberg in 1998.


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94


path. Everything from single particles such as

chromosomes up to an entire living organism, for

example Caenorhabditis elegans, is successfully

transported by applying LPC, thus protecting the

biological information or viability of the specimen.

At present, LMPC is the only published technology

able to microdissect and catapult viable cells as well

as tissues for further cultivation after isolation.

Furthermore, the isolation process can be

performed under sterile conditions. The processitself has no detrimental effects on isolated living

cells as they proliferate very well after being

catapulted. This opens a new approach, in

establishing homogeneous cell populations by

clonal expansion, to characterising cell types or

studying differentiation processes in developmental

biology. As the effective laser energy is

concentrated on a minute focal spot only, it is even

possible to perform laser microsurgery or

microinjection within living specimens without

affecting their viability. Numerous publications in

the fields of cell and developmental biology or from

assisted human fertilisation procedures have proventhe safety of 337nm nitrogen laser-based

microdissection and microsurgery.

S amp l e F e a tu r e s a nd

Spe c imen P r epa r a t i o n

Almost any biological sample is suitable for the non-

contact LMPC-technology. Depending on the nature

of the sample, catapulting may be performed directly,

as is the case for cytocentrifuged specimens, single

cells or homogeneous tissue areas. However, tissue

preparations are usually inhomogeneous and consist

of a mixture of different cell types. To avoid

contamination with unselected material, it is advisable

to perform laser microdissection prior to catapulting,

in order to obtain pure samples. An intuitive graphical

user interface software, the PALM RoboSoftware,

facilitates the selection of areas of interest. A wide

palette of drawing tools for marking the incision path

in preselection mode enables the outlining and

colour-coding of independent target areas all over the

entire slide or even from different slides, after serialsectioning. These selected target areas are listed in an

element list protocol, which allows target grouping

and experimental scheduling. The list of elements is

the main tool for summary and display of the outlined

samples and corresponding area measures, the colour-

dependent sorting of the outlined areas and laser

activation. Choosing from the coloured chart, the

computer will microdissect and/or catapult only

elements showing the particular preferred colour.

Thus, the laser cuts a clear gap between selected and

non-selected regions. Within the narrow laser cut, all

biological material is ablated and, therefore, thecaptured specimen is free of contamination. In the

immediate surroundings of the gap, cells and their

biological relevant content, like DNA, RNA or

proteins, remain intact and are perfectly suitable for

subsequent analyses. Specimens can be prepared

either directly onto glass slides or onto membrane-

mounted slides. The LPC membrane serves as a

backbone, which holds the selected tissue area close

together and facilitates the capture of larger tissue

areas or fragile, small samples. The laser first operates

around the selected area and cuts the tissue together

with the underlying membrane. With the catapult

shot of the laser, the entire selected area is ejected outof the object plane and catapulted directly into a

collection device, routinely filled with buffer or

Figure 1: Automated and Non-contact Laser Microdissection and

Pressure Catapulting Realised in the PALM MicroBeam HT

1.http://www.palm-microlaser.com/aboutus/PALM-Referenzen.pdf

Modern molecular biomedical research relies on the

capability of pure sample preparation.


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mineral oil. The comparison of samples catapulted

from membrane-mounted versus glass-mounted

tissue has shown that there is no difference in

subsequent molecular analysis.

Due to the fact that tissue sections for LMPC

cannot be routinely embedded and coverslipped,their morphology sometimes appears quite

different when compared with standard embedded

tissue sections. For improved, colour-balanced

contrast and resolution of specimens, PALM

Liquid CoverGlass N has proven to be the most

useful solution. Furthermore, this fluidic coverslip

preserves RNA integrity in tissue sections and

allows prolonged sample handling time. In

addition, PALM AdhesiveTubes provide further

improvement of balance contrast and resolution

and simultaneously allow harvesting of laser-

captured material without prior application of

buffer. This also prolongs the sample harvestingtime, by avoiding the danger of crystallisation

through the evaporation of salty buffers in the

collection device. Several protocols for sample

preparation and numerous downstream application

techniques have been developed during the past

few years. Special membrane-spanned object slides

for cell or tissue preparation (PALM

MembraneSlides), double-membrane culture dishes

for live cell capture (PALM

DuplexDish) andcustomised microfuge caps or microtiter plates for

improved or higher throughput laser capture were

developed and are available through P.A.L.M.

Microlaser Technologies AG.2

Au t o ma t e d C e l l R e c o g n i t i o n

The PALM MicroBeam System can optionally be

equipped with features for fluorescence microscopy.

Thus, LMPC is possible under simultaneous

fluorescence observation. The high degree of auto-

mation realised in the latest generation of PALM

MicroBeam HT Systems can optionally be augmented

by image-analysing software modules, allowingautomated fast scanning functions for specimen

identification and image processing. A further step

96

Sources: Tissue sections

Cytospins

Cell, blood or mucus smears

Vital cells from culture, mucus , saliva , blood, etc.

Preparation: Frozen or fixed

Embedded

Stained: HE-, MG-, PAP-,

immuno-staining, FISH, etc.

Precision:Capture of pure and

homogeneous samples

of any shape and size:

Large cell areas

Single cells

Subcellular Subcellular compounds

Automation: Image processing to find the

specimen under fluorescence

or brightfield illumination

Colour-coding, selection

throughout the slide or

serial sections

Automatic capture into

multi-cap strips or microtiter

plates or directly onto targets

such as chips and wafers

Specimen preparation

and Selection

LMPCfor pure

sample captureRNA

DNA

Protein

LMPC is the key technology for functional genomics,

transcriptomics and proteomics

Functional downstream analyses

Sequencing genes

Gene mutation

Single gene analysis

DNA-arrays

Messenger RNA

Transcript expression

Profiles

RNA-arrays

Post-translational

Protein modifications

Protein profiles

Protein arrays Eva

luation

ofresu

lts

data

mining

an

d

data

ware

housing

Table 1: Work Flow for Possible LMPC Applications

Key: HE = haematoxylin-eosin-stained; MG = Masson-Goldner-stained; PAP = Papanicolaou-stained; FISH = fluorescence in situ hybridisation.


2. http://www.palm-microlaser.com

Laser microdissection and micro-manipulation ... are

becoming more and more important for the understanding of

cellular physiology and pathology.


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towards automation is provided by a robotic

collection device, the PALM RoboMover, which

introduces a completely software-controlled sample

collection and allows for complex experimental

designs. This instrument enables the user to directly

catapult into a broad range of targets, such as multi-

cap strips or microtiter plates, microarray or biochip

wafers. The combination of these software modules

with the RoboMover and the highly precise

RoboStage II allows the MicroBeam HT System to

scan, detect, isolate and finally capture the specimen of

interest in a fully automated manner. Auto-marked

cells or cell areas are subsequently extracted

automatically by the appropriate laser functions. These

versatile, automated scanning software modules

provide the advantage of fast and reliable detection

and auto-evaluation of particular cells, cell

components or chromosomes, based on optimised

classifiers by means of morphological phenotypes.These efficient detection algorithms are trained by

P.A.L.M. to achieve integrated, interactive classifiers

for optimised recognition and accurate results.

P A L M M i c r o B e a m

A n O u t l o o k

Laser microdissection has provided scientists with

dramatically improved accuracy of histological and

cellular sampling over conventional retrieval

methods, resulting in a significant increase in the

specificity of downstream molecular analyses. Thepurity of sample preparation using LMPC is of

paramount importance for functional genomic and

proteomic studies. Microarray techniques based on

pure and homogeneous samples have a higher

potential for meaningful and valuable data and new

tumour markers may be identified that could not

be detected by using bulk material. Automated,

non-contact catapulting will save time, prevent the

danger of losing specimen during pipetting and

minimise contamination with unwanted material.

This highly sensitive and material-saving

technology is deeply crucial in, for example,cancer research, diagnosis of disease and in-patient-

tailored therapy, especially if only a small amount

of cells is available. Automated, high-throughput

LMPC, including software-based object

recognition and a robotic collection device,

enables rapid and even high-volume cellular

sampling. This high-speed collection device will

be beneficial and appreciated in routine laboratory

work, especially in microarray technologies, where

thousands of cells are required, as well as in

pharmaceutical drug screening and development.

In summary, this versatile laser micro-manipulation

and microdissection system is a state-of-the-art tool

for use throughout the entire field of modern

molecular research and medical analyses.

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