Abstract

A first-order thermal analysis is applied to Laser Capture Microdissection (LCM), a new microscope technique for routine targeting and extraction of specific cells from tissue sections for subsequent multiplex molecular analysis. In LCM a polymer film placed in contact with the tissue is focally activated by a pulsed IR laser beam and is melted and bonded to adjacent targeted cells. A three-dimensional finite-element model is used to predict the thermal transients within the polymer, the captured tissue, and its macromolecules. The simulations allow a comparison of models for the physical process of LCM with the experimental data on the dependence of the transfer spot size on laser power. The validated physical model and the thermal simulations permit optimization of the complex LCM parameter space for a wide variety of configurations and applications.

© 1998 Optical Society of America

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References

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  1. M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
    [CrossRef] [PubMed]
  2. R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).
  3. M. Webber, ed., CRC Handbook of Laser Science and Technology, Vol. 5: Optical Materials; Section 1.2. Applications: Materials for High Density Data Storage (CRC Press, Boca Raton, Fla., 1987), pp. 65–203.
  4. A. Marchant, Optical Recording, A Technical Overview (Addison-Wesley, Reading, Mass., 1990), pp. 353–360.
  5. C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).
  6. “Thermoplastics Materials Property and Price Chart,” in Plastics World (McGraw-Hill, New York, 1966).
  7. R. Perry, ed., Chemical Engineers Handbook, 5th ed. (McGraw-Hill, New York, 1973).
  8. A. Kirk, Packaging and Industrial Polymers Division, DuPont, Wilmington, Del. 19880 (personal communication, 1997).
  9. H. S. Carslaw, J. C. Jaeger, Conduction of Heat in Solids, 2nd ed. (Clarendon, Oxford, UK, 1988), pp. 1–10.
  10. Ref. 8, p. 264.
  11. J. P. Den Hartog, Strength of Materials (Dover, New York, 1961), p. 140.

1997 (1)

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

1996 (1)

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Bonner, R. F.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Carslaw, H. S.

H. S. Carslaw, J. C. Jaeger, Conduction of Heat in Solids, 2nd ed. (Clarendon, Oxford, UK, 1988), pp. 1–10.

Chuaqui, R. F.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Cole, K.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

Emmert-Buck, M. R.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Ghany, M.

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Goldstein, S. R.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Jaeger, J. C.

H. S. Carslaw, J. C. Jaeger, Conduction of Heat in Solids, 2nd ed. (Clarendon, Oxford, UK, 1988), pp. 1–10.

Kirk, A.

A. Kirk, Packaging and Industrial Polymers Division, DuPont, Wilmington, Del. 19880 (personal communication, 1997).

Liotta, L. A.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Marchant, A.

A. Marchant, Optical Recording, A Technical Overview (Addison-Wesley, Reading, Mass., 1990), pp. 353–360.

Peterson, J. I.

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Pohida, T.

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Smith, P. D.

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Suarez-Quian, C. A.

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Weiss, R. A.

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Wellner, E.

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

Zhuang, Z.

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

Science (2)

M. R. Emmert-Buck, R. F. Bonner, P. D. Smith, R. F. Chuaqui, Z. Zhuang, S. R. Goldstein, R. A. Weiss, L. A. Liotta, “Laser capture microdissection,” Science 274, 998–1001 (1996).
[CrossRef] [PubMed]

R. F. Bonner, M. R. Emmert-Buck, K. Cole, T. Pohida, R. F. Chuaqui, S. R. Goldstein, L. A. LiottaTech. Sight, “Laser capture microdissection: molecular analysis of tissue,” Science 278, 1481–1483 (1997).

Other (9)

M. Webber, ed., CRC Handbook of Laser Science and Technology, Vol. 5: Optical Materials; Section 1.2. Applications: Materials for High Density Data Storage (CRC Press, Boca Raton, Fla., 1987), pp. 65–203.

A. Marchant, Optical Recording, A Technical Overview (Addison-Wesley, Reading, Mass., 1990), pp. 353–360.

C. A. Suarez-Quian, S. R. Goldstein, T. Pohida, P. D. Smith, J. I. Peterson, E. Wellner, M. Ghany, R. F. Bonner, “Laser Capture Microdissection (LCM) of single cells from complex tissues,” BioTechniques (to be published).

“Thermoplastics Materials Property and Price Chart,” in Plastics World (McGraw-Hill, New York, 1966).

R. Perry, ed., Chemical Engineers Handbook, 5th ed. (McGraw-Hill, New York, 1973).

A. Kirk, Packaging and Industrial Polymers Division, DuPont, Wilmington, Del. 19880 (personal communication, 1997).

H. S. Carslaw, J. C. Jaeger, Conduction of Heat in Solids, 2nd ed. (Clarendon, Oxford, UK, 1988), pp. 1–10.

Ref. 8, p. 264.

J. P. Den Hartog, Strength of Materials (Dover, New York, 1961), p. 140.

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Figures (8)

Fig. 1
Fig. 1

Geometric configurations of different LCM systems: A, original CO2 laser LCM scheme in which the strong absorption of all thermoplastic polymers at 10.6 μm was utilized to prove feasibility on a variety of EVA polymer formulations. B, LCM design modified to use a near-IR laser-diode EVA polymer containing a dissolved near-IR-absorbing dye matched to a laser-diode wavelength and attached pressure plate. Plastic cap (pressure plate), tissue, and glass slide are all transparent and nonabsorbing of the near IR. C, prototype single-cell LCM epi design in which an imaging inverted microscope objective is used to focus the IR laser through the thin section onto the absorbing polymer with an attached pressure plate. In these figures L EVA and L glass are the thicknesses of the EVA film and glass, respectively; ϕlaser is the diameter of the laser beam; and CL is the axis of symmetry (center line of the beam).

Fig. 2
Fig. 2

Thermal transients in LCM from finite-element simulations: (a) Time dependence during a 0.6-s pulse at the center (r = 0) and edge (r = 50 μm) of the CO2 laser beam at each of three interfaces: - - - -, polymer–air (EVA); – – – –, polymer– tissue (Tissue); – –⋯– –, tissue–glass (Glass). (b) Radial profiles at the tissue–polymer interface for a CO2 laser LCM [(a) and Fig. 1A] at different times after the onset of the laser pulse. (c) Time dependence during a 50-ms pulse at the center (r = 0) and edge (r = 15 μm) of the laser beam (laser-diode LCM in Fig. 1B): - - - -, at the depth of maximum temperature in EVA (EVA); – – – –, at the polymer–tissue interface (Tissue); –·–, at the tissue–glass interface (Glass).

Fig. 3
Fig. 3

Radial profiles of the laser-diode LCM temperature rise at the EVA–tissue interface at the end of a 50-ms laser pulse. Nominal cases and indicated changes from nominal are shown: (a) condenser-side irradiation (Fig. 1B); (b) epi irradiation (Fig. 1C).

Fig. 4
Fig. 4

Axial profiles of the laser-diode LCM temperature rise on the z axis (r = 0). Nominal cases and indicated changes from nominal are shown: (a) condenser-side irradiation (Fig. 1B); (b) epi irradiation (Fig. 1C).

Fig. 5
Fig. 5

Radial profiles at the end of a 50-ms laser pulse of ITz. The normalized z integral of the laser-diode LCM temperature rises within the EVA [proportional to thermally induced axial displacement of the polymer surface as per the discussion above Eq. (9)]. Nominal cases and indicated changes from nominal are shown: (a) condenser-side irradiation (Fig. 1B); (b) epi irradiation (Fig. 1C).

Fig. 6
Fig. 6

EVA volume expansion (integral from 0 to r of 2πr times ITz from Fig. 5) at the end of a 50-ms laser pulse. Nominal cases and indicated changes from nominal are shown: (a) condenser-side irradiation (Fig. 1B); (b) epi irradiation (Fig. 1C).

Fig. 7
Fig. 7

Experimentally measured diameters (+) of the laser-diode LCM (Fig. 1B) transferred tissue as incident power was varied compared with three physical models for LCM fit when our thermal finite-element simulation results were used: ····, use of Eq. (8) to determine the maximum radius at which EVA at the tissue interface melts (fitted melting temperature, 66 °C); ┄, use of Eq. (10) to determine the maximum radius at which polymer axial thermal expansion is greater than the distance equal to the total air space between the resting polymer surface and the glass surface (fitted distance, 3.8 μm, when an a priori estimate of α = 0.00088/°C is used); ━, use of Eq. (11) to determine the radius at which the thermal volume expansion of EVA matches the total air space between the resting polymer surface and the glass surface at that radius (a priori estimates of α = 0.00088/°C, g = 1 μm, and γ = 0.67 are used). Numbers are predictions of temperature rise T p at the periphery of some of the experimental LCM transfers. Radial flow of excess expanded hot polymer is likely to increase the peripheral temperatures above these values that permit transfer.

Fig. 8
Fig. 8

Predicted laser-diode LCM transfer diameter versus incident power when a validated model from Eq. (11) for a 50-ms laser-diode pulse is used. Nominal cases and indicated changes from nominal are shown: (a) Condenser-side irradiation (Fig. 1B); (b) epi irradiation (Fig. 1C).

Tables (2)

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Table 1 Thermal and Optical Properties

Tables Icon

Table 2 Summary of Simulation Results

Equations (12)

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W i s = P in / A ε c   exp - ε cs   inside the top-hat radius , = 0   outside the top-hat radius ,
W i s = P ab / A 1 / 1 - 10 - OD 1 / λ exp - s / λ ,
1 / α T / τ = W i / k + 2 T / z 2 + 1 / r / r r T / r ,
T i = T i + 1 ,
k T / z i = - k T / z i + 1 ,
ρ c T / τ = rW i + k 2 T / z 2 + / r k T / r ,
ρ c T / τ = rW i + div k   grad   T ,
T = P / P n   *   T 0   *   T / T 0 + T amb ,
P = P n   *   T crit / T 0   *   T / T 0 ,
α = 1 - 2 / 3 / 1 + A   *   β ,
P = P n   *   Δ / α / IT 0   *   ITz / IT 0 ,
P = P n   *   γ h + g / α   *   π r t 2 / V c ,

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